Fc fusion proteins comprising novel linkers or arrangements

ABSTRACT

The application provides Fc fusion proteins having novel arrangements. In one embodiment, the application provides Fc fusion proteins comprising a  10 Fn3 domain. In another embodiment, the application provides Fc fusion proteins comprising linkers derived from the naturally occurring C-terminal tail regions of membrane bound or secretory immunoglobulins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Patent Application No. PCT/US2012/033665, filed Apr. 13,2012, which claims priority to U.S. Provisional Patent Application No.61/475,004, filed Apr. 13, 2011, the entire contents of which areincorporated in their entirety by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 10, 2015, isnamed MXI_526US_Sequence_Listing.txt and is 143,879 bytes in size.

BACKGROUND

The utility of many therapeutics, particularly biologicals such aspeptides, polypeptides and polynucleotides, suffer from inadequate serumhalf-lives. This necessitates the administration of such therapeutics athigh frequencies and/or higher doses, or the use of sustained releaseformulations, in order to maintain the serum levels necessary fortherapeutic effects. Frequent systemic administration of drugs isassociated with considerable negative side effects. For example,frequent systemic injections represent a considerable discomfort to thesubject, and pose a high risk of administration related infections, andmay require hospitalization or frequent visits to the hospital, inparticular when the therapeutic is to be administered intravenously.Moreover, in long term treatments daily intravenous injections can alsolead to considerable side effects of tissue scarring and vascularpathologies caused by the repeated puncturing of vessels. Similarproblems are known for all frequent systemic administrations oftherapeutics, such as, for example, the administration of insulin todiabetics, or interferon drugs in patients suffering from multiplesclerosis. All these factors lead to a decrease in patient complianceand increased costs for the health system.

One method for increasing the serum half-life of a protein is to attachit to a pharmacokinetic moiety. One type of pharmacokinetic moiety thathas been used is an “Fc” domain of an antibody. Antibodies comprise twofunctionally independent parts, a variable domain known as “Fab”, whichbinds antigen, and a constant domain known as “Fc”, which links to sucheffector functions as complement activation and attack by phagocyticcells. An Fc domain has a long serum half-life. Capon et al. (1989),Nature 337: 525-31. When fused to a therapeutic protein, an Fc domaincan provide longer half-life or incorporate such functions as Fcreceptor binding, protein A binding, complement fixation and perhapseven placental transfer.

This application provides novel Fc fusion proteins that increase theserum half-life of various therapeutics, polypeptides having increasedserum half-life, and methods for increasing the serum half-life oftherapeutics.

SUMMARY

The application provides novel Fc fusion proteins.

In one aspect, the application provides a polypeptide comprising: (a) a¹⁰Fn3 domain having an altered amino acid sequence relative to thewild-type sequence, wherein the ¹⁰Fn3 domain binds to a target moleculewith a K_(D) of less than 500 nM; (b) an immunoglobulin (Ig) Fc domain;and (c) a hinge sequence.

In certain embodiments, the polypeptide may have the followingarrangement from N-terminus to C-terminus: ¹⁰Fn3 domain-hinge-Fc domain.In alternative embodiments, the polypeptide may have the followingarrangement from N-terminus to C-terminus: hinge-Fc domain-linker-¹⁰Fn3domain.

In exemplary embodiments, the polypeptide is a dimer. The dimerpreferably forms via a disulfide bond between free cysteine residues inthe hinge region.

In certain embodiments, the polypeptide further comprises a second ¹⁰Fn3domain having an altered amino acid sequence relative to the wild-typesequence and wherein the second ¹⁰Fn3 domain binds to a target moleculewith a K_(D) of less than 500 nM. The two ¹⁰Fn3 domains may bind to thesame or different targets.

In certain embodiments, the Fc domain of the polypeptide may be from anIgG, IgM, IgD, IgE, or IgA. In exemplary embodiments, the Fc domain isderived from an IgG, such as an IgG1.

In various embodiments, the hinge sequence and the Fc domain may bederived from the same or different Ig isotypes.

In certain embodiments, the hinge region comprises residues 104-119 ofSEQ ID NO: 22 or a sequence having at least 90% sequence identitythereto.

In another aspect, the application provides a polypeptide comprising animmunoglobulin Fc domain and a heterologous polypeptide, wherein theheterologous polypeptide is fused to the C-terminus of the Fc domain bya polypeptide linker comprising a sequence derived from the C-terminaltail region of the heavy chain of a membrane bound or secretoryimmunoglobulin.

In certain embodiments, the polypeptide linker comprises a sequence thatis at least 80% identical to any one of SEQ ID NOs: 51-70, comprises atleast 5 or 10 contiguous amino acids of any one of SEQ ID NOs: 51-70, orcomprises the sequence of any one of SEQ ID NOs: 51-70.

In certain embodiments, the heterologous polypeptide comprises a ¹⁰Fn3domain. In certain embodiments, the heterologous polypeptide comprisestwo ¹⁰Fn3 domains, wherein the two ¹⁰Fn3 domains may bind to the same ordifferent targets.

In another aspect, the application provides a nucleic acid encoding theFc fusion proteins provided herein. Also provided are vectors, includingexpression vectors, comprising a nucleic acid encoding any of the Fcfusion proteins described herein. Also provided are host cellscontaining such expression vectors and methods for producing the Fcfusion proteins described herein in the host cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Inhibition of PCSK9:EGFA (left panel) and PCSK9:ATI-972 (rightpanel) by PRD460 in a FRET assay.

FIG. 2. Inhibition of PCSK9-induced LDLR depletion from HepG2 cellsurface by anti-PCSK9 Adnectins.

FIG. 3. Inhibition of PCSK9-AF647 cell entry in HepG2 cells byanti-PCSK9 Adnectins.

FIG. 4. Plasma unbound hPCSK9 levels in transgenic mice treated withPRD460 (dosed i.p.).

FIG. 5. Effect of PRD460 (15 mg/kg i.v.) on LDL-C and free PCSK9 incynomolgus monkeys (mean+/−SEM, n=3).

FIG. 6. Pharmacokinetics of PRD460 and ATI-1081 following intravenousadministration into cynomolgus monkeys.

FIG. 7. Pharmacokinetics of PRD460 and Adn-1 following intravenousadministration into cynomolgus monkeys.

FIG. 8. Pharmacokinetics of C7FLFc, Adn-2 and Adn-3 followingintravenous administration into cynomolgus monkeys.

FIG. 9. Pharmacokinetics of Adn-1 in cynomolgus monkeys followingintravenous and subcutaneous administration.

FIG. 10. Pharmacokinetics of PRD460, PRD461, PRD239, PRD713, Adn-1,Adn-4, Adn-5, Adn-6 and Adn-7 following intravenous administration intomice.

FIG. 11. Pharmacokinetics of C7FLFc, Adn-8, Adn-3 and Adn-9 followingintravenous administration into mice.

FIG. 12. Pharmacokinetics of PRD239 and PRD713 following intravenousadministration into mice.

FIG. 13. Pharmacokinetics of PRD460 following intravenous administrationinto C57B1/6 and nude mice.

FIG. 14. Pharmacokinetics of PRD460 and PRD461 following intravenousadministration into mice.

FIG. 15. Inhibition of BaF3 proliferation by C7FL-Fc.

FIG. 16. Inhibition of PCSK9-induced LDLR depletion from HepG2 cellsurface by anti-PCSK9 Fc-¹⁰Fn3 fusion proteins.

FIG. 17. Inhibition of PCSK9-induced LDLR depletion from HepG2 cellsurface by anti-PCSK9 Fc-¹⁰Fn3 fusion proteins.

FIG. 18. Average yield of high-throughput mammalian expressed Fc-¹⁰Fn3proteins.

FIG. 19. Monomer score of high-throughput mammalian expressed Fc-¹⁰Fn3proteins.

FIG. 20. Average yield of mid-scale expressed Fc-¹⁰Fn3 proteins.

FIG. 21. Monomer score of mid-scale expressed Fc-¹⁰Fn3 proteins.

FIG. 22. LC-MS data of mid-scale expressed Fc-¹⁰Fn3 proteins.

FIG. 23. DSC data of mid-scale expressed Fc-¹⁰Fn3 proteins.

FIG. 24. SPR sensogram data for the binding of mid-scale expressedFc-¹⁰Fn3 proteins to target.

FIG. 25. Comparison of the wild type human □1 constant region Fc (Fc1)amino acid sequence (SEQ ID NO: 154) with Fc variants Fc4 through Fc23(SEQ ID NOS: 155-173, respectively). The C_(H)1 domain of the human □1constant region is not part of the Fc and is therefore not shown. Thelocations of the hinge region, the C_(H)2 domain, and the C_(H)3 domainare indicated. The Cys residues normally involved in disulfide bondingto the light chain constant region (LC) and heavy chain constant region(HC) are indicated. A “.” indicates identity to wild type at thatposition. A “-” indicates a gap introduced into the sequence to optimizealignment. Only locations where the Fc variants differ from wild typeare shown, otherwise the Fc sequences match the wild type sequenceshown. The sequence positions are numbered according to the universallyaccepted EU Index numbering system for immunoglobulin proteins. ***indicates the location of the carboxyl terminus and is included toclarify the difference in the carboxyl terminus of Fc6 relative to theother Fc versions.

FIG. 26. Comparison of the wild type BALB/c mouse □2a constant region Fc(mFc1)(SEQ ID NO: 174) and the wild type C57BL/6 mouse □2c constantregion Fc (mFc3)(SEQ ID NO: 176) amino acid sequences with mouse Fceffector function minus variants mFc2 (SEQ ID NO: 175) and mFc4 (SEQ IDNO: 177). The location of the hinge region, the C_(H)2 domain, and theC_(H)3 domain are indicated. The Cys residues normally involved indisulfide bonding to the heavy chain constant region (HC) are indicated.A “.” indicates identity to wild type at that position. A “-” indicatesa gap inserted in the sequence to maximize the alignment. The sequencepositions are numbered according to the universally accepted EU Indexnumbering system for immunoglobulin proteins.

FIG. 27. Immunogenicity of 1571G04-PEG in cynomolgus monkeys.

FIG. 28. Immunogenicity of 1571G04-Fc in cynomolgus monkeys.

DETAILED DESCRIPTION Definitions

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

The notations “mpk”, “mg/kg”, or “mg per kg” refer to milligrams perkilogram. All notations are used interchangeably throughout the presentdisclosure.

The “half-life” of a polypeptide can generally be defined as the timetaken for the serum concentration of the polypeptide to be reduced by50%, in vivo, for example due to degradation of the polypeptide and/orclearance or sequestration of the polypeptide by natural mechanisms. Thehalf-life can be determined in any manner known per se, such as bypharmacokinetic analysis. Suitable techniques will be clear to theperson skilled in the art, and may, for example, generally involve thesteps of administering a suitable dose of a polypeptide to a rodent orprimate; collecting blood samples or other samples from said primate atregular intervals; determining the level or concentration of thepolypeptide in said blood sample; and calculating, from (a plot of) thedata thus obtained, the time until the level or concentration of thepolypeptide has been reduced by 50% compared to the initial level upondosing. Methods for determining half-life may be found, for example, inKenneth et al., Chemical Stability of Pharmaceuticals: A Handbook forPharmacists (1986); Peters et al, Pharmacokinete analysis: A PracticalApproach (1996); and “Pharmacokinetics”, M Gibaldi & D Perron, publishedby Marcel Dekker, 2nd Rev. edition (1982).

Half-life can be expressed using parameters such as the t1/2-alpha,t1/2-beta, HL_Lambda_z, and the area under the curve (AUC). In thepresent specification, an “increase in half-life” refers to an increasein any one of these parameters, any two of these parameters, any threeof these parameters or all four of these parameters. An “increase inhalf-life” in particular refers to an increase in the t1/2-beta and/orHL_Lambda_z, either with or without an increase in the t1/2-alpha and/orthe AUC or both. Other PK parameters that can be assessed include volumeof distribution (VD), clearance (CL), and mean residence time (MRT). Inthe present specification, a “change in pharmacokinetics” refers tochanges in any one of these parameters, any two of these parameters, orall three of these parameters, in the presence or absence of changes inthe half-life parameters listed above.

Fc Fusion Proteins

This application relates to novel Fc fusion proteins having improvedproperties. The application provides Fc-X fusion proteins having novellinkers that confer favorable properties such as increased expression,reduced immunogenicity and/or increased protease resistance. Theapplication also relates to novel fibronectin based scaffold polypeptideFc fusions having improved pharmacokinetics properties compared to theirnon-Fc fusion counterparts. The novel fibronectin based scaffoldpolypeptide Fc fusions described herein may be designed to bind to anytarget of interest. In exemplary embodiments, the target is an antigen,a polypeptide or a therapeutic protein target of interest. Exemplarytherapeutically desirable targets, include, for example, tumor necrosisfactor alpha (TNF-alpha), delta-like protein 4 (DLL4), interleukin 17(IL-17), proprotein convertase subtilisin kexin type 9 (PCSK9), pregnaneX receptor (PXR), epidermal growth factor receptor (EGFR), insulin-likegrowth factor 1 receptor (IGF-1R), vascular endothelial growth factorreceptor (VEGFR2) and interleukin 23 (IL-23).

Fc-X Fusion Proteins with Novel Linkers

In many cases, Fc fusion proteins having the arrangement Fc-X (e.g., aheterologous polypeptide attached to the C-terminus of the Fc domain)contain a linker sequence separating the immunoglobulin domain (Igdomain) from the heterologous polypeptide. These linkers typically areartificial flexible domains, such as GGGGS. However, these sequences arenot natural sequences and may lead to undesirable properties, such asimmunogenicity. Accordingly, in one aspect, the application provides fornovel, improved Fc fusion proteins using linker sequences derived fromnaturally occurring antibody sequences, including natural allelic orsplice variants. In particular, the application provides novel Fc fusionproteins having the arrangement from N-terminus to C-terminus: Fc-L₁-X,where Fc is an Fc domain (as described further below), L₁ is linker asequence derived from the natural tail sequence of a membrane-bound orsecretory form of an antibody, and X is a heterologous polypeptide. Thelinker will be positioned in the Fc fusion protein in its naturalcontext, e.g., in its natural place in the Ig CH3 of CH4 sequence. Thesenatural linker sequences will permit the construction of Fc fusionproteins with linkers of varying length that will be in a naturalcontext and therefore likely to have favorable properties with regard toexpression, immunogenicity and/or protease resistance.

Most immunoglobulins exist in soluble and membrane-bound isoforms. Themembrane-bound isoform consists of the soluble form with a tailalternatively spliced in the CH3 or CH4 domain towards the C-terminusbefore the stop codon. The tail of the membrane-bound isoform consistsof a linker, a trans-membrane segment, and an intracellular segment.Certain immunoglobulins, such as IgA, contain tail segments in theirsecretory forms, which may also be used as linkers.

In one embodiment, the application provides an Fc fusion protein havingthe arrangement Fc-L₁-X, wherein L₁ is a linker sequence derived fromthe tail segment of a membrane bound form of an immunoglobulin.Exemplary linker sequences include for example: (i) the tail region ofthe membrane long isoform of IgA1 (mα1_(L)):SCSVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 51), (ii) the tail region ofthe membrane variant long isoform of IgA1 (mα1_(L) with extra cys):SCCVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 52), (iii) the tail regionof the membrane short isoform of IgA1 (mα1_(s) with 6 amino acidN-terminal deletion): DWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 53), (iv)the tail region of the membrane bound form of IgA2:SCCVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 54), (v) the tail region ofthe membrane bound form of IgD: YLAMTPLIPQSKDENSDDYTTFDDVGS (SEQ ID NO:55), (vi) the tail region of the membrane-bound form of IgE:ELDVCVEEAEGEAPW (SEQ ID NO: 56), (vii) the tail region of the membranebound form of IgG: ELQLEESCAEAQDGELDG (SEQ ID NO: 57), and (viii) thetail region of the membrane bound form of IgM EGEVSADEEGFEN (SEQ ID NO:58).

In other embodiments, the application provides the application providesan Fc fusion protein having the arrangement Fc-L₁-X, wherein L1 is alinker sequence derived from the tail segment of a secretory or solubleform of an immunoglobulin. Exemplary linker sequences include forexample: (i) the tail region of the soluble form of IgA1:KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 59), (ii) the tail region of the solubleform of IgA2: KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 60), (iii) the tail regionof the soluble form of IgD: YVTDHGPMK (SEQ ID NO: 61), and (iv): thetail region of the soluble form of IgM: PTLYNVSLVMSDTAGTCY (SEQ ID NO:62).

In certain embodiments, it may be desirable to have a linker sequencecontaining a free cysteine residue in order to permit the formation of adisulfide bond between linkers thereby forming dimers of the Fc fusionproteins. In other embodiments, it may be desirable to alter the linkersequences to remove free cysteine residues, e.g., by mutating one ormore cysteine residues in a linker to another residue, such as a serine,alanine or glycine. Examples of linker sequences derived from the tailregions of membrane bound immunoglobulins that have been altered toremove free cysteine residues include: (i)SXSVADWQMPPPYVVLDLPQETLEEETPGAN, wherein X is serine, alanine or glycine(SEQ ID NO: 63), (ii) SXXVADWQMPPPYVVLDLPQETLEEETPGAN, wherein each X isindependently selected from serine, alanine or glycine (SEQ ID NO: 64),(iii) SXXVADWQMPPPYVVLDLPQETLEEETPGAN, wherein each X is independentlyselected from serine, alanine or glycine (SEQ ID NO: 65), (iv)ELDVXVEEAEGEAPW, wherein X is serine, alanine or glycine (SEQ ID NO:66), and (v) ELQLEESXAEAQDGELDG, wherein X is serine, alanine or glycine(SEQ ID NO: 67). Examples of linker sequences derived from the tailregions of secretory forms of immunoglobulins that have been altered toremove free cysteine residues include: (i) KPTHVNVSVVMAEVDGTXY, whereinX is serine, alanine or glycine (SEQ ID NO: 68), (ii)KPTHVNVSVVMAEVDGTXY, wherein X is serine, alanine or glycine (SEQ ID NO:69), and (iii) PTLYNVSLVMSDTAGTXY, wherein X is serine, alanine orglycine (SEQ ID NO: 70).

In one embodiment, the application provides an Fc fusion protein havingthe arrangement Fc-L₁-X, wherein L₁ is a linker sequence comprising,consisting essentially of, or consisting of an amino acid sequence thatis at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% toany one of SEQ ID NOs: 51-70, or an amino acid sequence comprising,consisting essentially of, or consisting of any one of SEQ ID NOs:51-70. In another embodiment, the application provides an Fc fusionprotein having the arrangement Fc-L₁-X, wherein L₁ is a linker sequencecomprising at least 2, 5, 10, 12, 15, 20, 25, or 30 contiguous aminoacid residues from any of SEQ ID NOs: 51-70, or a sequence comprisingfrom 1-5, 1-10, 1-15, 1-20, 1-25, 2-5, 2-10, 2-15, 2-20, 2-25, 5-10,5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30,20-25, 25-30 or 25-30 contiguous amino acid residues from any of SEQ IDNOs: 51-70. In certain embodiments, the linker sequence does not containa cysteine residue. In certain embodiments, the linker sequence may beextended in length by repetition, concatenation or combination of anyone of SEQ ID NOs: 51-70, or fragments thereof.

In certain embodiments, the Fc-L₁-X fusion proteins provided herein mayhave increased expression, decreased immunogenicity, and/or improvedprotease resistance relative to Fc fusion proteins having differentlinker sequences. For example, a host cell comprising an expressionvector encoding for an Fc-L₁-X fusion protein provided herein mayprovide at least 10%, 20%, 30%, 40%, 50% 75% or 100% greater expressionthan an equivalent Fc fusion protein having a non-naturally occurringlinker sequence, or at least 2-fold, 3-fold, 4-fold, 5-fold or 10-foldhigher levels of expression than an equivalent Fc fusion protein havinga non-naturally occurring linker sequence. In certain embodiments, anFc-L₁-X fusion protein provided herein may have reduced immunogenicityrelative an equivalent Fc fusion protein having a non-naturallyoccurring linker sequence. The immunogenicity of a polypeptide describedherein may be assessed, for example, by one or more of the followingmethods: Human Leukocyte Antigen (“HLA”) binding, in silico predictionof HLA binding (for example, with the Epimatrix program), in vitroactivation of human T-cells, in vivo animal immune response, or othermethods for evaluating immunogenicity potential. In other embodiments,an Fc-L₁-X fusion protein provided herein may have increased proteaseresistance relative to an equivalent Fc fusion protein having anon-naturally occurring linker sequence.

The Fc-L₁-X fusion proteins described herein contain an X portion thatmay be any protein of interest. In exemplary embodiments, the X portionis a therapeutic peptide or protein, such as, for example, interferonalpha, L-asparaginas, or granulocyte colony-stimulating factor. Incertain embodiments, the X portion of the fusions described herein is anantibody, or fragment thereof, such as, for example, and anti-TNF-alphaantibody. In an exemplary embodiment, the X portion of the Fc fusionproteins is a polypeptide comprising ¹⁰Fn3 domain, including, forexample, a polypeptide comprising a ¹⁰Fn3 domain that binds to a targetsuch as tumor necrosis factor alpha (TNF-alpha), delta-like protein 4(DLL4), interleukin 17 (IL-17), proprotein convertase subtilisin kexintype 9 (PCSK9), pregnane X receptor (PXR), epidermal growth factorreceptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R),vascular endothelial growth factor receptor (VEGFR2) and interleukin 23(IL-23).

Fibronectin Based Scaffold Protein-Fc Fusions

Provided herein are Fc fusion proteins comprising an Fc domain fused toa polypeptide that binds to a target. The polypeptide that binds to atarget may be derived from a fibronectin or tenascin molecule or it maybe a synthetic molecule that is based on the sequences and structure offibronectin and tenascin molecules. Polypeptides that may be used in Fcfusion proteins are described, e.g., in WO2010/051274, WO2010/051310 andWO2009/086116.

In one aspect, the application provides Fc fusion proteins comprising anFc domain fused, a polypeptide comprising a ¹⁰Fn3 domain, and a hingesequence. These fusions are referred to collectively herein as Fc-¹⁰Fn3fusions. The Fc-¹⁰Fn3 fusion proteins may be arranged in either order,e.g., from N-terminus to C-terminus, Fc-¹⁰Fn3 or ¹⁰Fn3-Fc. In anexemplary embodiment, a Fc-¹⁰Fn3 fusion protein has the followingarrangement from N-terminus to C-terminus: ¹⁰Fn3-hinge-Fc domain,wherein ¹⁰Fn3 refers to a polypeptide comprising a ¹⁰Fn3 domain, hingerefers to an immunoglobulin hinge sequence as described further herein,and Fc refers to an immunoglobulin Fc domain. In an exemplaryembodiment, a Fc-¹⁰Fn3 fusion protein has the following arrangement fromN-terminus to C-terminus: ¹⁰Fn3-Fc domain, wherein ¹⁰Fn3 refers to apolypeptide comprising a ¹⁰Fn3 domain and Fc refers to an immunoglobulinFc domain. In another exemplary embodiment, a Fc-¹⁰Fn3 fusion proteinhas the following arrangement from N-terminus to C-terminus: hinge-Fcdomain-L₂-¹⁰Fn3, wherein hinge refers to an immunoglobulin hingesequence as described further herein, Fc refers to an immunoglobulin Fcdomain, L₂ refers to a linker as further defined herein, and ¹⁰Fn3refers to a polypeptide comprising a ¹⁰Fn3 domain. In an exemplaryembodiment, a Fc-¹⁰Fn3 fusion protein has the following arrangement fromN-terminus to C-terminus: Fc domain-L₂-¹⁰Fn3, wherein Fc refers to animmunoglobulin Fc domain, L₂ refers to a linker as further definedherein, and ¹⁰Fn3 refers to a polypeptide comprising a ¹⁰Fn3 domain. Inan exemplary embodiment, a Fc-¹⁰Fn3 fusion protein has the followingarrangement from N-terminus to C-terminus: Fc domain-¹⁰Fn3, wherein Fcrefers to an immunoglobulin Fc domain and ¹⁰Fn3 refers to a polypeptidecomprising a ¹⁰Fn3 domain. In an exemplary embodiment, a Fc-¹⁰Fn3 fusionprotein has the following arrangement from N-terminus to C-terminus:hinge-Fc domain-¹⁰Fn3, wherein hinge refers to an immunoglobulin hingesequence as described further herein, Fc refers to an immunoglobulin Fcdomain, and ¹⁰Fn3 refers to a polypeptide comprising a ¹⁰Fn3 domain. Ineither orientation, the Fc-¹⁰Fn3 fusion proteins described herein mayfurther contain an N-terminal methionine and/or a leader sequence (e.g.,for expression in mammalian cells).

In certain embodiments, the Fc-¹⁰Fn3 fusion proteins described hereincomprise a hinge sequence, preferably a hinge sequence that contains afree cysteine residue that is capable of forming a disulfide bond suchthat the Fc-¹⁰Fn3 fusion protein forms a dimer. The hinge sequence maynaturally contain a cysteine residue, or may be engineered to containone or more cysteine residues.

The Fc-¹⁰Fn3 fusion proteins described herein may contain animmunoglobulin hinge region. The hinge region may be derived fromantibodies belonging any of the immunoglobulin classes, i.e. IgA, IgD,IgE, IgG, or IgM. In certain embodiments, the hinge region is derivedfrom any of the IgG antibody subclasses, i.e. IgG1, IgG2, IgG3, andIgG4. In some embodiments, the hinge region may further include residuesderived from the CH1 and CH2 regions that flank the core hinge sequence,as discussed further below.

Shown below is the sequence of a human IgG1 immunoglobulin constantregion, and the relative position of each domain within the constantregion are indicated based on the EU numbering format:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22). The core hinge sequenceis underlined, and the CH1 region is italicized; the CH2 and CH3 regionsare in regular text. It should be understood that the C-terminal lysineis optional. In certain embodiments, the C-terminal lysine of an IgGsequence may be removed or replaced with a non-lysine amino acid, suchas alanine, to further increase the serum half-life of the Fc fusionprotein.

In certain embodiments, the Fc-¹⁰Fn3 fusion proteins described hereincomprise a hinge region derived from a human IgG1. In some embodiments,the hinge region comprises the core hinge residues spanning positions104-119 of SEQ ID NO: 22 (DKTHTCPPCPAPELLG; SEQ ID NO: 23) of IgG1,which corresponds to positions 221-236 according to EU numbering.

In certain embodiments, the hinge sequence may include substitutionsthat confer desirable pharmacokinetic, biophysical, and/or biologicalproperties. Some exemplary hinge sequences includeEPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 24; core hinge region underlined),EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 25; core hinge region underlined),EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 26; core hinge region underlined),DKTHTCPPCPAPELLGGPS (SEQ ID NO: 27; core hinge region underlined), andDKTHTCPPCPAPELLGGSS (SEQ ID NO: 28, core hinge region underlined). Inone embodiment, the hinge sequence is a derivative of an IgG1 hingecomprising a P122S substitution based on the numbering in SEQ ID NO: 22(EU numbering 238) (e.g., the Proline residue at position 122 in SEQ IDNO: 22 is substituted with serine). The P122S substitution ablates Fceffector function and is exemplified by the hinges having any one of SEQID NOs: 25, 26, and 28. In another embodiment, the hinge sequence is aderivative of an IgG1 hinge comprising D104G and K105S substitutionsbased on the numbering in SEQ ID NO: 22 (EU numbering 221-222). TheD104G and K105S substitutions remove a potential cleavage site andtherefore increase the protease resistance of the fusion molecule. Ahinge having D104G and K105S substitutions is exemplified in SEQ ID NO:26. In another embodiment, the hinge sequence is a derivative of an IgG1hinge comprising a C103S substitution based on the numbering in SEQ IDNO: 22 (EU numbering 220). The C103S substitution prevents impropercysteine bond formation in the absence of a light chain. Hinges having aC103S substitution are exemplified by SEQ ID NOs: 24-26.

In one embodiment, the application provides a Fc-¹⁰Fn3 fusion protein,wherein the hinge sequence comprises, consists essentially of, orconsists of an amino acid sequence that is at least 50%, 60%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% to any one of SEQ ID NOs: 24-28, orcomprises, consists essentially of, or consists of an amino acidsequence of any one of SEQ ID NOs: 24-28. In another embodiment, theapplication provides a Fc-¹⁰Fn3 fusion protein, wherein the hingeportion comprises at least 2, 5, 10, 12, 15, 18 or 20 contiguous aminoacid residues from any of SEQ ID NOs: 24-28, or a sequence comprisingfrom 1-5, 1-10, 1-15, 1-20, 2-5, 2-10, 2-15, 2-20, 5-10, 5-15, 5-20,10-15, 10-20, or 15-20 contiguous amino acid residues from any of SEQ IDNOs: 24-28. In exemplary embodiments, the hinge sequence comprises acysteine residue.

In certain embodiments, an Fc fusion protein does not comprise a hinge.For example, an Fc fusion protein may comprise an Fc domain linked to aheterologous protein, e.g., in the Fc-X or X-Fc format, withoutcomprising a hinge or a core hinge. In one example, an Fc fusion proteindoes not comprise the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 89) or avariant thereof.

In certain embodiments, an Fc fusion protein does not comprise a linker.For example, an Fc fusion protein may comprise an Fc domain that islinked directly to a heterologous protein, e.g., a ¹⁰Fn3 protein withoutan intervening sequence. In certain embodiments, there may be 1, 2, 3, 4or 5 amino acids (e.g., from 1-5 or 1-10 amino acids) between the Fcdomain and the heterologous protein. Such Fc fusion proteins may be X-Fcor Fc-X fusion proteins, wherein X is the heterologous protein, andwherein X and Fc are directly linked to each other.

In certain embodiments, an Fc fusion protein does not comprise a hingeand does not comprise a linker.

The Fc-¹⁰Fn3 fusion proteins described herein comprise an Fc domain, asdescribed further below. In certain embodiments, the Fc domain and thehinge region may be derived from one antibody class or subclass. Forexample, the hinge region and the Fc domain may be derived from IgG1. Inother embodiments, the Fc domain and hinge region may be derived fromdifferent antibody classes or subclasses. For example, the Fc domain maybe derived from IgG2 or IgG4 and the hinge region may be derived fromIgG1.

In certain embodiments, a Fc-¹⁰Fn3 fusion protein described herein hasthe arrangement hinge-Fc domain-L₂-¹⁰Fn3, wherein L₂ is a linker thatconnects the Fc domain to the polypeptide comprising a ¹⁰Fn3 domain. Inexemplary embodiments, the L₂ linker is selected from the groupconsisting of: GSGSGSGSGSGS (SEQ ID NO: 33), AGGGGSG (SEQ ID NO: 37),AGGGGSGG (SEQ ID NO: 38), QPDEPGGS (SEQ ID NO: 45), ELQLEESAAEAQDGELD(SEQ ID NO: 46), TVAAPS (SEQ ID NO: 47), QPDEPGGSG (SEQ ID NO: 48),ELQLEESAAEAQDGELDG (SEQ ID NO: 49), TVAAPSG (SEQ ID NO: 50), and any oneof SEQ ID NOs: 51-70, 81-88 and 90-98. In other embodiments, the L₂linker comprises, consists essentially of, or consists of an amino acidsequence that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% to any one of SEQ ID NOs: 33, 37-38, 45-70, 81-88 and 90-98,or comprises, consists essentially of, or consists of any one of SEQ IDNOs: 33, 37-38, 45-70, 81-88 and 90-98. In another embodiment, L₂comprises at least 2, 5, 10, 12, 15, 20, 25, or 30 contiguous amino acidresidues from any of SEQ ID NOs: 33, 37-38, 45-70, 81-88 and 90-98, or asequence comprising from 1-5, 1-10, 1-15, 1-20, 1-25, 2-5, 2-10, 2-15,2-20, 2-25, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30,15-20, 15-25, 15-30, 20-25, 25-30 or 25-30 contiguous amino acidresidues from any of SEQ ID NOs: 33, 37-38, 45-70, 81-88 and 90-98. Incertain embodiments, the L₂ linker sequence does not contain a cysteineresidue. In certain embodiments, the linker sequence may be extended inlength by repetition, concatenation or combination of any one of SEQ IDNOs: 33, 37-38, 45-70, 81-88 and 90-98, or fragments thereof.

Suitable Fc domains and polypeptides comprising ¹⁰Fn3 domains for use inthe Fc-¹⁰Fn3 fusion proteins are described further below.

In certain embodiments, the Fc-¹⁰Fn3 fusion proteins provided herein mayhave an increased serum half-life relative to a ¹⁰Fn3 domain without theFc fusion or relative to a ¹⁰Fn3 domain fused to a differentpharmacokinetic moiety, such as, for example a polyethylene glycol (PEG)moiety. For example, a Fc-¹⁰Fn3 fusion protein provided herein may havea serum half life that is at least 10%, 20%, 30%, 40%, 50% 75% or 100%greater than the serum half life of an equivalent ¹⁰Fn3 domain withoutthe Fc domain or relative to an equivalent ¹⁰Fn3 domain fused to adifferent pharmacokinetic moiety, such as, for example a polyethyleneglycol (PEG) moiety. In certain embodiments, a Fc-¹⁰Fn3 fusion proteinprovided herein has a serum half life that is at least 2-fold, 3-fold,4-fold, 5-fold or 10-fold longer than the serum half life of anequivalent ¹⁰Fn3 domain without the Fc domain or relative to anequivalent ¹⁰Fn3 domain fused to a different pharmacokinetic moiety,such as, for example a polyethylene glycol (PEG) moiety.

In certain embodiments, an Fc fusion protein, e.g., a ¹⁰Fn3-Fc fusionprotein, is a dimer, wherein each monomer comprises a fusion protein (ahomodimer). In certain embodiments, an Fc fusion protein, e.g., a¹⁰Fn3-Fc fusion protein, is a heterodimer comprising, e.g., a monomerthat comprises an Fc fusion protein and a monomer that comprises an Fcthat is not linked to a heterologous protein. The Fc portion of amonomer may comprise one or more amino acid modifications (or mutations)relative to a wild type Fc that favor dimer formation with another Fc.For example, an Fc of a dimer may comprise a “hole” and the other Fc ofthe dimer may comprise a “bump” or “knob,” as described, e.g., inWO96/027011; U.S. Pat. No. 5,731,168 and U.S. Pat. No. 5,821,333. Othermodification, such as electrostatic modifications may be used to enhancedimer formation. Exemplary modifications are described, e.g., inWO2007/110205; WO2009/089004 and WO2010/129304. Such changes areparticularly useful for enhancing the association of two heterologousmonomers to form a dimer, such as a dimer that comprises a monomercomprising an Fc fusion protein and a monomer comprising an Fc that isdifferent from the Fc fusion protein, e.g., by the lack of aheterologous protein. Monomers of the dimer may be linked covalently ornon covalently to each other.

In certain embodiments, an Fc fusion protein comprises a monomercomprising the structure X-Fc and a monomer comprising the structureFc-X (or Fc-Y), wherein each monomer may optionally comprise a linkerand optionally comprise a hinge.

A heterodimeric Fc fusion protein may comprise a single chain Fc (scFc),wherein the first and the second Fc domain (or the first and the secondhinge-Fc domains) are linked through a linker. In one embodiment, a scFccomprises in N- to C-terminal order a first CH2 domain, which first CH2domain is linked to a first CH3 domain, which CH3 domain is linked to anFc linker, which Fc linker is linked the a second CH2 domain, whichsecond CH2 domain is linked to a second CH3 domain, wherein the firstand the second CH2 and CH3 domains associate to form a dimeric Fc. AnscFc may comprise in N- to C-terminal order a first hinge, which firsthinge is linked to a first CH2 domain, which first CH2 domain is linkedto a first CH3 domain, which first CH3 domain is linked to an Fc linker,which Fc linker is linked to a second hinge, which second hinge islinked to a second CH2 domain, which second CH2 domain is linked to asecond CH3 domain, wherein the first and the second hinges, CH2 domainsand CH3 domains associate to form a dimeric Fc. scFcs are described,e.g., in WO2008/131242, WO2008/143954 and WO2008/012543.

Fc Domains

Described herein are polypeptide fusions that comprise an Fc portionfused to a heterologous portion. In some aspects, the heterologousportion is a ¹⁰Fn3 domain.

As used herein, “Fc portion” encompasses domains derived from theconstant region of an immunoglobulin, preferably a human immunoglobulin,including a fragment, analog, variant, mutant or derivative of theconstant region. Suitable immunoglobulins include IgG1, IgG2, IgG3,IgG4, and other classes such as IgA, IgD, IgE and IgM. The constantregion of an immunoglobulin is defined as a naturally-occurring orsynthetically-produced polypeptide homologous to the immunoglobulinC-terminal region, and can include a CH1 domain, a hinge, a CH2 domain,a CH3 domain, or a CH4 domain, separately or in combination.

The constant region of an immunoglobulin is responsible for manyimportant antibody functions including Fc receptor (FcR) binding andcomplement fixation. There are five major classes of heavy chainconstant region, classified as IgA, IgG, IgD, IgE, IgM, each withcharacteristic effector functions designated by isotype. For example,IgG is separated into four subclasses known as IgG1, IgG2, IgG3, andIgG4.

Ig molecules interact with multiple classes of cellular receptors. Forexample IgG molecules interact with three classes of Fcγreceptors (FcγR)specific for the IgG class of antibody, namely FcγRI, FcγRII, andFcγRIII. The important sequences for the binding of IgG to the FcγRreceptors have been reported to be located in the CH2 and CH3 domains.The serum half-life of an antibody is influenced by the ability of thatantibody to bind to an Fc receptor (FcR). Similarly, the serum half-lifeof IgFc fusion proteins is also influenced by the ability to bind tosuch receptors (Gillies S D et al., (1999) Cancer Res. 59:2159-66).

The fusion proteins disclosed herein comprise an Fc portion thatincludes at least a portion of the carboxy-terminus of an immunoglobulinheavy chain. For example, the Fc portion may comprise: a CH2 domain, aCH3 domain, a CH4 domain, a CH2-CH3 domain, a CH2-CH4 domain, aCH2-CH3-CH4 domain, a hinge-CH2 domain, a hinge-CH2-CH3 domain, ahing-CH2-CH4 domain, or a hinge-CH2-CH3-CH4 domain. The Fc domain may bederived from antibodies belonging any of the immunoglobulin classes,i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses,i.e., IgG1, IgG2, IgG3, and IgG4. The Fc domain may be a naturallyoccurring Fc sequence, including natural allelic or splice variants.Alternatively, the Fc domain may be a hybrid domain comprising a portionof an Fc domain from two or more different Ig isotypes, for example, anIgG2/IgG4 hybrid Fc domain. In exemplary embodiments, the Fc domain isderived from a human immunoglobulin molecule. Alternatively, the Fcdomain may be a humanized or deimmunized version of an Fc domain from anon-human animal, including but not limited to mouse, rat, rabbit,camel, llama, dromedary and monkey.

In certain embodiments, the Fc domain is a variant Fc sequence, e.g., anFc sequence that has been modified (e.g., by amino acid substitution,deletion and/or insertion) relative to a parent Fc sequence (e.g., anunmodified Fc polypeptide that is subsequently modified to generate avariant), to provide desirable structural features and/or biologicalactivity.

For example, one may make modifications in the Fc region in order togenerate an Fc variant that (a) has increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) has increased ordecreased affinity for C1q and/or (d) has increased or decreasedaffinity for a Fc receptor relative to the parent Fc. Such Fc regionvariants will generally comprise at least one amino acid modification inthe Fc region. Combining amino acid modifications is thought to beparticularly desirable. For example, the variant Fc region may includetwo, three, four, five, etc substitutions therein, e.g. of the specificFc region positions identified herein.

A variant Fc domain may also comprise a sequence alteration whereinsites involved in disulfide bond formation are removed. Such removal mayavoid reaction with other cysteine-containing proteins present in thehost cell used to produce the molecules of the invention. For thispurpose, the cysteine-containing segment at the N-terminus may betruncated or cysteine residues may be deleted or substituted with otheramino acids (e.g., alanyl, seryl). Even when cysteine residues areremoved, the single chain Fc domains can still form a dimeric Fc domainthat is held together non-covalently. In other embodiments, a native Fcdomain may be modified to make it more compatible with a selected hostcell. For example, one may remove the PA sequence near the N-terminus ofa typical native Fc, which may be recognized by a digestive enzyme in E.coli such as proline iminopeptidase. One may also add an N-terminalmethionine residue, especially when the molecule is expressedrecombinantly in a bacterial cell such as E. coli. In anotherembodiment, a portion of the N-terminus of a native Fc domain is removedto prevent N-terminal heterogeneity when expressed in a selected hostcell. For this purpose, one may delete any of the first 20 amino acidresidues at the N-terminus, particularly those at positions 1, 2, 3, 4and 5. In other embodiments, one or more glycosylation sites within theFc domain may be removed. Residues that are typically glycosylated(e.g., asparagine) may confer cytolytic response. Such residues may bedeleted or substituted with unglycosylated residues (e.g., alanine) Inother embodiments, sites involved in interaction with complement, suchas the C1q binding site, may be removed from the Fc domain. For example,one may delete or substitute the EKK sequence of human IgG1. In certainembodiments, sites that affect binding to Fc receptors may be removed,preferably sites other than salvage receptor binding sites. In otherembodiments, an Fc domain may be modified to remove an ADCC site. ADCCsites are known in the art; see, for example, Molec. Immunol. 29 (5):633-9 (1992) with regard to ADCC sites in IgG1. Specific examples ofvariant Fc domains are disclosed for example, in WO 97/34631 and WO96/32478.

In certain embodiments, an Fc fusion protein described herein comprisesthe CH2 and CH3 regions of a human IgG1 as shown below:VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31). It should beunderstood that the glycine and lysine at the end of SEQ ID NO: 31 areoptional. In other embodiments, an Fc fusion protein described hereincomprises an Fc domain that is at least 50%, 60%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31. In otherembodiments, an Fc fusion protein described herein comprises an Fcdomain having at least 50, 100, or 150 contiguous amino acids of SEQ IDNO: 31. In other embodiments, an Fc fusion protein described hereincomprises an Fc domain having from 50-100, 50-150, or 100-150 contiguousamino acids of SEQ ID NO: 31. In yet other embodiments, an Fc fusionprotein described herein comprises an Fc domain comprising SEQ ID NO: 31with from 1-5, 1-10, 1-15, 1-20, or 1-25 substitutions or conservativesubstitutions.

Additional Fc variants are described below. It is understood that the Fcregions of the disclosure comprise the numbering scheme according to theEU index as in Kabat et al. (1991, NIH Publication 91-3242, NationalTechnical Information Service, Springfield, Va.).

The present disclosure encompasses variant Fc portions which havealtered binding properties for an Fc ligand relative to an unmodifiedparent Fc molecule. For example, an Fc fusion protein described hereinmay comprise an Fc region having one or more of amino acid residues 234,235, 236, 237, 297, 318, 320 and 322 substituted to a different aminoacid residue, such that the variant Fc region has an altered affinityfor an effector ligand, e.g., an Fc receptor or the C1 component ofcomplement, as described in U.S. Pat. Nos. 5,624,821 and 5,648,260, bothto Winter et al.

In another example, one or more of amino acid residues 329, 331 and 322can be replaced such that the variant Fc region has altered C1q bindingand/or reduced or abolished complement dependent cytotoxicity (CDC), asdescribed in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 may be altered to thereby alter the ability of thevariant Fc region to fix complement. This approach is described furtherin WO 94/29351 by Bodmer et al.

In yet another example, the Fc region may be modified to increaseantibody dependent cellular cytotoxicity (ADCC) and/or to increase theaffinity for an Fey receptor by modifying one or more amino acids at thefollowing positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245,247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322,324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340,360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434,435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D,239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variantsinclude 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F,267E/324T, and 267E/268F/324T. Other modifications for enhancing FcyRand complement interactions include but are not limited to substitutions298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L,292P, 300L, 396L, 3051, and 396L. These and other modifications arereviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

Fc modifications that increase binding to an Fc gamma receptor includeamino acid modifications at any one or more of amino acid positions 238,239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272,279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301,303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360,373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437,438 or 439 of the Fc region, wherein the numbering of the residues inthe Fc region is that of the EU index as in Kabat (WO00/42072).

Other Fc modifications that can be made to Fcs are those for reducing orablating binding to FcyR5 and/or complement proteins, thereby reducingor ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.Exemplary modifications include but are not limited substitutions,insertions, and deletions at positions 234, 235, 236, 237, 267, 269,325, and 328, wherein numbering is according to the EU index. Exemplarysubstitutions include but are not limited to 234G, 235G, 236R, 237K,267R, 269R, 325L, and 328R, wherein numbering is according to the EUindex. An Fc variant may comprise 236R/328R. Other modifications forreducing FcyR and complement interactions include substitutions 297A,234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S,229S, 238S, 233P, and 234V, as well as removal of the glycosylation atposition 297 by mutational or enzymatic means or by production inorganisms such as bacteria that do not glycosylate proteins. These andother modifications are reviewed in Strohl, 2009, Current Opinion inBiotechnology 20:685-691.

Optionally, the Fc region may comprise a non-naturally occurring aminoacid residue at additional and/or alternative positions known to oneskilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publications WO00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114).

Fc variants that enhance affinity for an inhibitory receptor FcyRllb mayalso be used. Such variants may provide an Fc fusion protein withimmunomodulatory activities related to FcyRl lb⁺ cells, including forexample B cells and monocytes. In one embodiment, the Fc variantsprovide selectively enhanced affinity to FcyRllb relative to one or moreactivating receptors. Modifications for altering binding to FcyRl lbinclude one or more modifications at a position selected from the groupconsisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327,328, and 332, according to the EU index. Exemplary substitutions forenhancing FcyRl lb affinity include but are not limited to 234D, 234E,234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M,267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D,268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding toFcyRl lb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D,267E/268E, and 267E/328F.

The affinities and binding properties of an Fc region for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art including but not limitedto, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay(ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACOREanalysis), and other methods such as indirect binding assays,competitive inhibition assays, fluorescence resonance energy transfer(FRET), gel electrophoresis and chromatography (e.g., gel filtration).These and other methods may utilize a label on one or more of thecomponents being examined and/or employ a variety of detection methodsincluding but not limited to chromogenic, fluorescent, luminescent, orisotopic labels. A detailed description of binding affinities andkinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4thEd., Lippincott-Raven, Philadelphia (1999), which focuses onantibody-immunogen interactions.

An Fc fusion protein of the present disclosure may also comprise an Fcportion which increases the serum half-life of the Fc-fusion protein.For example, this may be done by increasing the binding affinity of theFc region for FcRn. For example, one or more of more of followingresidues can be mutated: 252, 254, 256, 433, 435, 436, as described inU.S. Pat. No. 6,277,375.

Other exemplary variants that increase binding to FcRn and/or improvepharmacokinetic properties include substitutions at positions 259, 308,428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H,434F, 434Y, and 434M. Other variants that increase Fc binding to FcRninclude: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J.Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A,378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry,2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q,256E, 256D, 256T, 309P, 311 S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F,434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall Acqua et al.Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006,Journal of Biological Chemistry 281:23514-23524). Other modificationsfor modulating FcRn binding are described in Yeung et al., 2010, JImmunol, 182:7663-7671. In certain embodiments, hybrid IgG isotypes withparticular biological characteristics may be used. For example, an1gG1/1gG3 hybrid variant may be constructed by substituting 1gG1positions in the CH2 and/or CH3 region with the amino acids from 1gG3 atpositions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more substitutions,e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R,and 436F. In other embodiments of the invention, an 1gG1/1gG2 hybridvariant may be constructed by substituting IgG2 positions in the CH2and/or CH3 region with amino acids from 1gG1 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody may be constructedthat comprises one or more substitutions, e.g., one or more of thefollowing amino acid substitutions: 233E, 234L, 235L, -236G (referringto an insertion of a glycine at position 236), and 327A.

In certain embodiments, the glycosylation of the Fc is modified.Oligosaccharides that are covalently attached to the Fc region can bechanged, for example by expressing an IgG in various organisms or celllines, engineered or otherwise (for example Lec-13 CHO cells or rathybridoma YB2/0 cells), by regulating enzymes involved in theglycosylation pathway (for example FUT8 [a1,6-fucosyltranserase] and/orβ1-4-N-acetylglucosaminyltransferase III [GnTIII]), by modifyingcarbohydrate(s) after the IgG has been expressed, or by expressing an Fcfusion protein in the presence of fucose analogs as enzymaticinhibitors. Other methods for modifying glycoforms of Fc fusion proteinsinclude using glycoengineered strains of yeast (Li et al., 2006, NatureBiotechnology 24(2):210-215), moss (Nechansky et al., 2007, Mol.Immunol. 44(7): 1826-8), and plants (Cox et al., 2006, Nat Biotechnol24(12):1591-7). In one embodiment, Fc fusions are glycoengineered toalter the level of sialylation. Higher levels of sialylated Fc glycansin Fc molecules can adversely impact functionality (Scallon et al.,2007, Mol Immunol. 44(7): 1524-34), and differences in levels of Fcsialylation can result in modified anti-inflammatory activity (Kaneko etal., 2006, Science 313:670-673). The level of glycosylation of an Fcmolecule may also be modified by specific mutations. For example, amutation at amino acid position 297 or 299 removes the glycosyation atposition 297. Such mutants may also be used with Fc fusion proteins.

Other Fc modifications that may be used in Fc fusion proteins includethose described in WO88/07054, WO88/07089, U.S. Pat. No. 6,277,375,WO99/051642, WO01/058957, WO2003/074679, WO2004/029207, U.S. Pat. No.7,317,091 and WO2004/099249.

Moreover, the following Fc variants may also be used for the Fc portionof the Fc fusion proteins described herein. FIG. 25 shows the comparisonof the wild type human γ1 constant region Fc (human IgG1Fc; designatedas Fc1 in FIG. 25) with Fc4 (SEQ ID NO: 99), Fc5 (SEQ ID NO: 100), Fc6(SEQ ID NO: 101), Fc7 (SEQ ID NO: 102), Fc8 (SEQ ID NO: 103), Fc9 (SEQID NO: 104), Fc10 (SEQ ID NO: 105), Fc11 (SEQ ID NO: 106), Fc12 (SEQ IDNO: 107), Fc13 (SEQ ID NO: 108), Fc14 (SEQ ID NO: 109), Fc15 (SEQ ID NO:110), Fc16 (SEQ ID NO: 111), Fc17 (SEQ ID NO: 112), Fc18 (SEQ ID NO:113), Fc19 (SEQ ID NO: 114), Fc21 (SEQ ID NO: 115), Fc22 (SEQ ID NO:116), Fc23 (SEQ ID NO: 117). In some aspects, an Fc fusion proteindescribed herein comprises an Fc domain having at least 50, 100, or 150contiguous amino acids of any one of SEQ ID NOs: 99-117. In otherembodiments, an Fc fusion protein described herein comprises an Fcdomain having from 50-100, 50-150, or 100-150 contiguous amino acids ofSEQ ID NOs: 99-117. In yet other embodiments, an Fc fusion proteindescribed herein comprises an Fc domain comprising SEQ ID NOs: 99-117with from 1-5,1-10, 1-15, 1-20, or 1-25 substitutions or conservativesubstitutions. The human wild type γ1 constant region sequence was firstdescribed by Leroy Hood's group in Ellison et al., Nucl. Acids Res.10:4071 (1982). EU Index positions 356, 358, and 431 define the G1m γ1haplotype. The wild type sequence shown here is of the G1m(1), positions356 and 368, and nG1m(2), position 431, haplotype.

The Fc4 variant contains a γ1 hinge region, but Arg 218 has beenintroduced in the hinge region to include a Bg1II restriction enzymerecognition sequence to facilitate cloning. Cys 220 is the Cys residuethat forms the disulfide bond to the light chain constant region in anintact immunoglobulin IgG1 protein. Fc4 also includes a Ser for Cysresidue substitution to prevent deleterious effects due to the potentialpresence of an unpaired sulfhydral group. The CH2 region of Fc4 is basedon the γ1 CH2 and contains three amino acid substitutions that reduce Fcγ receptor I (FcγRI) binding. These are the substitutions at EU indexpositions 234, 235, and 237. These substitutions were described by GregWinter's group in Duncan et al., Nature 332:563 (1988) and were shown inthat paper to reduce binding to the Fc γ RI.

Two amino acid substitutions in the complement C1q binding site wereintroduced to reduce complement fixation. These are the substitutions atEU index positions 330 and 331. The importance, or relevance, ofpositions 330 and 331 in complement C1q binding (or lack of complementfixation or activation) is described by Sherie Morrison's group in Taoet al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp.Med. 173:1483 (1991). The CH3 region in the Fc4 variant remainsidentical to the wild type γ1 Fc.

Fc5 is a variant of Fc4. In the Fc5 hinge region the Arg 218substitution was returned to the wild type Lys 218 residue. Fc5 containsthe same Cys 220 to Ser substitution as described above for Fc4. Fc5contains the same CH2 substitutions as does Fc4, and the Fc5 CH2 regionis identical to the wild type γ1 Fc.

The Fc6 variant contains the same hinge region substitutions as Fc5 andcontains the same CH2 substitutions as Fc4. The Fc6 CH3 region does notcontain a carboxyl terminal lysine residue. This particular Lys residuedoes not have an assigned EU index number. This lysine is removed to avarying degree from mature immunoglobulins and therefore predominantlynot found on circulating antibodies. The absence of this residue onrecombinant Fc fusion proteins may result in a more homogeneous product.

The Fc7 variant is identical to the wild type γ1 Fc in the hinge region.Its CH2 region is based on γ1 CH2, but the N-linked carbohydrateattachment site at residue Asn-297 is changed to Gln to produce adeglycosylated Fc. (See e.g., Tao and Morrison (1989) J. Immunol.143:2595-2601). The CH3 region is identical to the wild type γ1 Fc.

Fc8 variant has a hinge region that is identical to Fc4, and both theCH2 region and the CH3 region are identical to the corresponding wildtype γ1 Fc regions.

The Fc9 variant contains a shortened γ1 hinge starting at the Aspresidue just carboxy-terminal to the Cys residue involved in disulfidelinkage to the light chain. The remaining hinge sequence is identical tothe wild type γ1 hinge. Both the CH2 region sequence and the CH3 regionsequence are identical to the corresponding regions for the wild-type γ1Fc.

The Fc10 variant contains the same hinge region substitution as Fc5.Both the CH2 region sequence and the CH3 region sequence are identicalto the corresponding regions for the wild-type γ1 Fc.

The Fc11 variant contains the same hinge region substitutions as Fc5.Its CH2 domain is based on γ1 CH2, but contains the substitutions todecrease Fcγ Receptor binding (substitutions at EU index positions 234,235, and 237). Fc11 is wild type for C1q binding and complementfixation. The CH3 domain of Fc11 is identical to the wild type γ1 CH3.

The Fc12 variant contains a γ1 hinge with Cys 220 Ser, Cys 226 Ser, andCys 229 Ser substitutions, has a CH2 domain that is identical to that ofFc5, and has wild-type γ1 CH3 domain.

The Fc13 variant contains a γ1 hinge with Cys 220 Ser, Cys 226 Ser, andCys 229 Ser substitutions, has CH2 domain that is identical to that ofFc5, and has a wild-type γ1 CH3 with Tyr 407 Gly substitution.

The Fc14 variant contains a □1 hinge with Cys 220 Ser, Cys 226 Ser, andCys 229 Ser substitutions, has a wild-type □1 CH2, and has a wild-type□1 CH3 with Tyr 407 Gly substitution. The Fc15 variant contains a □4hinge with a Ser 228 Pro substitution to decrease IgG4 “Fab exchange”,and has a wild-type □4 CH2 and CH3 domains.

The Fc16 variant contains a γ1 hinge that contains a Cys 220 Sersubstitution, has a CH2 domain identical to the γ1 CH2, and has a CH3domain identical to the wild type γ4 CH3.

The Fc17 variant contains a γ1 hinge with a Cys 220 Ser substitution,has a γ1 CH2 domain with a Phe 243 Ala substitution, and has a CH3domain identical to the wild type γ1 CH3.

The Fc18 variant contains a γ1 hinge with a Cys 220 Ser substitution,has a γ1 CH2 domain identical to the wild type γ1 CH2, and contains a γ1CH3 with a H is 435 Ala substitution.

The Fc19 variant contains a hinge identical to Fc5, has a CH2 domainidentical to Fc5, except N-linked carbohydrate attachment site atresidue Asn-297 is changed to Gln to produce a deglycosylated Fc, andhas a CH3 domain identical to the wild type γ1 CH3.

The Fc21 variant contains a γ1 hinge with Cys 220 Ser, Cys 226 Ser, andCys 229 Ser substitutions, has a CH2 domain identical to Fc5, and has aγ1 CH3 with Phe 405 Ala and Tyr 407 Gly substitutions.

The Fc22 variant contains a γ1 hinge with Cys 220 Ser, Cys 226 Ser, andCys 229 Ser substitutions, has a CH2 domain identical to Fc1, and has aγ1 CH3 with Phe 405 Ala and Tyr 407 Gly substitutions.

The Fc23 variant contains a γ1 hinge with Cys 220 Ser substitution, hasa γ1 CH2 domain with Leu 234 Ala, Leu 235 Glu, Pro 331 Sersubstitutions, and a CH3 domain identical to the wild type γ1 Fc.

FIG. 26 shows an alignment of additional Fc variants that may also beused for the Fc portion of the Fc fusion proteins described herein. FIG.26 shows the comparison of the amino acid sequences of wild type BALB/cmouse γ2a constant region Fc (mFc1; SEQ ID NO: 118) and wild typeC57BL/6 mouse γ2c constant region Fc (mFc3; SEQ ID NO: 119) with twomouse Fc variants, mFc2 (SEQ ID NO: 120) and mFc4 (SEQ ID NO: 121),which have little or no effector function. The wild type C57BL/6γ2c wasinitially isolated and sequenced in the early 1980's and referred to asthe mouse γ2a, b allotype (Schreier et al. PNAS 78:4495 (1981)).Subsequent sequence analysis comparisons have shown that the genecorresponds in fact to mouse γ2c (Fukui et al., J. Mol. Cell. Immunol.1:321 (1984) and Morgado et al., EMBO J. 8:3245 (1989)). Note thatseveral different allotypes do exist for both the γ2a and γ2c sequences.The sequence of mFc1 corresponds to GenBank Accession #V00825 while thesequence of mFc3 corresponds to GenBank Accession #Y10606.

In some aspects, an Fc fusion protein described herein comprises an Fcdomain having at least 50, 100, or 150 contiguous amino acids of any oneof SEQ ID NOs: 118-121. In other embodiments, an Fc fusion proteindescribed herein comprises an Fc domain having from 50-100, 50-150, or100-150 contiguous amino acids of SEQ ID NOs: 118-121. In yet otherembodiments, an Fc fusion protein described herein comprises an Fcdomain comprising SEQ ID NOs: 118-121 with from 1-5, 1-10, 1-15, 1-20,or 1-25 substitutions or conservative substitutions.

The mFc1 variant contains a wild type BALB/c mouse γ2a Fc.

The mFc2 variant contains a BALB/c mouse γ2a hinge with a Gly 219 Sersubstitution. The mFc2 CH2 domain contains an amino acid substitutionrelative to mouse wild type γ2a at position 235 (Leu to Glu) toinactivate binding to FcγRI and FcγRII as described in Duncan et al.,Nature 332:563 (1988) and Zheng et al., J. Immunol. 163:4041 (1999).Three additional changes were made at the complement C1q binding site toreduce complement fixation at positions 318, 320 and 322. Thesesubstitutions are also described by Zheng et al. The interaction of IgGand C1q was originally identified in Duncan and Winter, Nature 332:738(1988). The CH3 domain is identical to the wild type mouse γ2a Fc.

The mFc3 variant contains a wild type C57BL/6 mouse γ2c Fc.

The mFc3 variant is identical to mFc3 except that it contains the Gly219 Ser and Leu 235 Glu substitutions present in mFc2.

Other modifications/substitutions/additions/deletions of the Fc domainwill be readily apparent to one skilled in the art.

Polypeptides Comprising ¹⁰Fn3 Domains

In certain embodiments, the Fc fusion proteins provided herein comprisea ¹⁰Fn3 domain, which is a fibronectin based scaffold protein.Fibronectin based scaffold proteins generally make use of a scaffoldderived from a fibronectin type III (Fn3) or Fn3-like domain andfunction in a manner characteristic of natural or engineered antibodies(that is, polyclonal, monoclonal, or single-chain antibodies) and, inaddition, possess structural advantages. Specifically, the structure ofthese antibody mimics has been designed for optimal folding, stability,and solubility, even under conditions that normally lead to the loss ofstructure and function in antibodies. An example of fibronectin-basedscaffold proteins are Adnectins™ (Adnexus, a wholly owned subsidiary ofBristol-Myers Squibb). Fibronectin-based scaffold proteins andAdnectins™ may be monovalent or multivalent.

An Fn3 domain is small, monomeric, soluble, and stable. It lacksdisulfide bonds and, therefore, is stable under reducing conditions. Theoverall structure of Fn3 resembles the Ig fold. Fn3 domains comprise, inorder from N-terminus to C-terminus, a beta or beta-like strand, A; aloop, AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-likestrand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a betaor beta-like strand, E; a loop, EF; a beta or beta-like strand, F; aloop, FG; and a beta or beta-like strand, G. The seven antiparallelβ-strands are arranged as two beta sheets that form a stable core, whilecreating two “faces” composed of the loops that connect the beta orbeta-like strands. Loops AB, CD, and EF are located at one face andloops BC, DE, and FG are located on the opposing face. Any or all ofloops AB, BC, CD, DE, EF and FG may participate in ligand binding. Thereare at least 15 different modules of Fn3, and while the sequencehomology between the modules is low, they all share a high similarity intertiary structure.

The amino acid sequence of the naturally occurring human tenthfibronectin type III domain, i.e., the tenth module of human Fn3(¹⁰Fn3), is set forth in SEQ ID NO: 1:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO:1) (the AB, CD and EFloops are underlined, and the BC, FG, and DE loops are emphasized inbold).

In SEQ ID NO:1, the AB loop corresponds to residues 15-16, the BC loopcorresponds to residues 21-30, the CD loop corresponds to residues39-45, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 60-66, and the FG loop corresponds to residues76-87. See e.g., Xu et al., Chemistry & Biology 2002 9:933-942. The BC,DE and FG loops align along one face of the molecule (sometimes referredto as the “north pole” loops) and the AB, CD and EF loops align alongthe opposite face of the molecule (sometimes referred to as the “southpole” loops). In SEQ ID NO: 1, beta strand A corresponds to residues9-14, beta strand B corresponds to residues 17-20, beta strand Ccorresponds to residues 31-38, beta strand D corresponds to residues46-50, beta strand E corresponds to residues 57-59, beta strand Fcorresponds to residues 67-75, and beta strand G corresponds to residues88-94. The strands are connected to each other through the correspondingloop, e.g., strands A and B are connected via loop AB in the formationof strand A, loop AB, strand B, etc. The first 8 amino acids of SEQ IDNO:1 (italicized above) may be deleted while still retaining bindingactivity of the molecule. Residues involved in forming the hydrophobiccore (the “core amino acid residues”) include the amino acidscorresponding to the following amino acids of SEQ ID NO: 1: L8, V10,A13, L18, I20, W22, Y32, I34, Y36, F48, V50, A57, I59, L62, Y68, I70,V72, A74, I88, I90 and Y92, wherein the core amino acid residues arerepresented by the single letter amino acid code followed by theposition at which they are located within SEQ ID NO: 1. See e.g.,Dickinson et al., J. Mol. Biol. 236: 1079-1092 (1994).

¹⁰Fn3 domains are structurally and functionally analogous to antibodies,specifically the variable region of an antibody. While ¹⁰Fn3 domains maybe described as “antibody mimics” or “antibody-like proteins”, they dooffer a number of advantages over conventional antibodies. Inparticular, they exhibit better folding and thermostability propertiesas compared to antibodies, and they lack disulphide bonds, which areknown to impede or prevent proper folding under certain conditions.

The BC, DE, and FG loops of ¹⁰Fn3 domains are analogous to thecomplementary determining regions (CDRs) from immunoglobulins.Alteration of the amino acid sequence in these loop regions changes thebinding specificity of ¹⁰Fn3. ¹⁰Fn3 domains with modifications in theAB, CD and EF loops may also be made in order to produce a molecule thatbinds to a desired target. The protein sequences outside of the loopsare analogous to the framework regions from immunoglobulins and play arole in the structural conformation of the ¹⁰Fn3. Alterations in theframework-like regions of ¹⁰Fn3 are permissible to the extent that thestructural conformation is not so altered as to disrupt ligand binding.Methods for generating ¹⁰Fn3 ligand specific binders have been describedin PCT Publication Nos. WO 00/034787, WO 01/64942, and WO 02/032925,disclosing high affinity TNFα binders, PCT Publication No. WO2008/097497, disclosing high affinity VEGFR2 binders, and PCTPublication No. WO 2008/066752, disclosing high affinity IGFIR binders.Additional references discussing ¹⁰Fn3 binders and methods of selectingbinders include PCT Publication Nos. WO 98/056915, WO 02/081497, and WO2008/031098 and U.S. Publication No. 2003186385.

As described above, amino acid residues corresponding to residues 21-30,51-56, and 76-87 of SEQ ID NO: 1 define the BC, DE and FG loops,respectively. However, it should be understood that not every residuewithin the loop region needs to be modified in order to achieve a ¹⁰Fn3binder having strong affinity for a desired target. For example, in manycases, only residues corresponding to amino acids 23-30 of the BC loopand 52-55 of the DE loop are modified and result in high affinity ¹⁰Fn3binders. Accordingly, in certain embodiments, the BC loop may be definedby amino acids corresponding to residues 23-30 of SEQ ID NO: 1, and theDE loop may be defined by amino acids corresponding to residues 52-55 ofSEQ ID NO: 1. Additionally, insertions and deletions in the loop regionsmay also be made while still producing high affinity ¹⁰Fn3 binders.

Accordingly, in some embodiments, one or more loops selected from BC,DE, and FG may be extended or shortened in length relative to thecorresponding loop in wild-type human ¹⁰Fn3. In some embodiments, thelength of the loop may be extended by 2-25 amino acids. In someembodiments, the length of the loop may be decreased by 1-11 aminoacids. In particular, the FG loop of ¹⁰Fn3 is 12 residues long, whereasthe corresponding loop in antibody heavy chains ranges from 4-28residues. To optimize antigen binding, therefore, the length of the FGloop of ¹⁰Fn3 may be altered in length as well as in sequence to coverthe CDR3 range of 4-28 residues to obtain the greatest possibleflexibility and affinity in antigen binding. In some embodiments, theintegrin-binding motif “arginine-glycine-aspartic acid” (RGD), locatedat residues 79-81 of SEQ ID NO: 1, may be modified in order to disruptintegrin binding. For example, the RGD sequence may be replaced with SGEor RGE.

As described herein, the non-ligand binding sequences of ¹⁰Fn3, i.e.,the “¹⁰Fn3 scaffold”, may be altered provided that the ¹⁰Fn3 retainsligand binding function and/or structural stability. In someembodiments, one or more of Asp 7, Glu 9, and Asp 23 are replaced byanother amino acid, such as, for example, a non-negatively charged aminoacid residue (e.g., Asn, Lys, etc.). These mutations have been reportedto have the effect of promoting greater stability of the mutant ¹⁰Fn3 atneutral pH as compared to the wild-type form (See, PCT Publication No.WO 02/04523). A variety of additional alterations in the ¹⁰Fn3 scaffoldthat are either beneficial or neutral have been disclosed. See, forexample, Batori et al., Protein Eng. 2002 15(12):1015-20; Koide et al.,Biochemistry 2001 40(34):10326-33. In some embodiments, the hydrophobiccore amino acids are not modified relative to the wild-type sequence. Inother embodiments, the following hydrophobic amino acids may be mutated:W22 and/or L62.

The ¹⁰Fn3 scaffold may be modified by one or more conservativesubstitutions. As many as 5%, 10%, 20% or even 30% or more of the aminoacids in the ¹⁰Fn3 scaffold may be altered by a conservativesubstitution without substantially altering the affinity of the ¹⁰Fn3for a ligand. In certain embodiments, the scaffold may comprise anywherefrom 0-15, 0-10, 0-8, 0-6, 0-5, 0-4, 0-3, 1-15, 1-10, 1-8, 1-6, 1-5,1-4, 1-3, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-15, or 5-10 conservativeamino acid substitutions. In certain embodiments, the substitutions inthe scaffold do not include substitutions of the hydrophobic core aminoacid residues. Preferably, the scaffold modification reduces the bindingaffinity of the ¹⁰Fn3 binder for a ligand by less than 100-fold,50-fold, 25-fold, 10-fold, 5-fold, or 2-fold. It may be that suchchanges will alter the immunogenicity of the ¹⁰Fn3 in vivo, and wherethe immunogenicity is decreased, such changes will be desirable. As usedherein, “conservative substitutions” refers to replacement of one aminoacid with another amino acid that is physically or functionally similarto the amino acid being replaced. That is, a conservative substitutionand its reference residue have similar size, shape, electric charge,chemical properties including the ability to form covalent or hydrogenbonds, or the like. Preferred conservative substitutions are thosefulfilling the criteria defined for an accepted point mutation inDayhoff et al., Atlas of Protein Sequence and Structure 5:345-352 (1978& Supp.). Examples of conservative substitutions are substitutionswithin the following groups: (a) valine, glycine; (b) glycine, alanine;(c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e)asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine,methionine; and (h) phenylalanine, tyrosine.

In some embodiments, the application provides an Fc fusion proteincomprising a ¹⁰Fn3 domain, wherein the ¹⁰Fn3 polypeptide is at least40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identity to thehuman ¹⁰Fn3 domain having the amino acid sequence of SEQ ID NO: 1. Muchof the variability will generally occur in one or more of the loops.Each of the beta or beta-like strands of a ¹⁰Fn3 domain in a fibronectinbased scaffold protein may comprise, consist essentially of, or consistof an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100%identical to the sequence of a corresponding beta or beta-like strand ofSEQ ID NO: 1, provided that such variation does not disrupt thestability of the polypeptide in physiological conditions. In exemplaryembodiments, the ¹⁰Fn3 domain binds to a desired target with a K_(D) ofless than 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 100 pM or less. Inexemplary embodiments, the fibronectin based scaffold protein bindsspecifically to a target that is not bound by a wild-type ¹⁰Fn3 domain,particularly the wild-type human ¹⁰Fn3 domain.

In some embodiments, the application provides an Fc fusion proteincomprising a ¹⁰Fn3 domain, wherein the ¹⁰Fn3 polypeptide has an aminoacid sequence at least 80, 85, 90, 95, 98, or 100% identical to thenon-loop regions of SEQ ID NO: 1, wherein at least one loop selectedfrom BC, DE, and FG is altered. In some embodiments, the altered BC loophas up to 10 amino acid substitutions, up to 4 amino acid deletions, upto 10 amino acid insertions, or a combination thereof. In someembodiments, the altered DE loop has up to 6 amino acid substitutions,up to 4 amino acid deletions, up to 13 amino acid insertions, or acombination thereof. In some embodiments, the FG loop has up to 12 aminoacid substitutions, up to 11 amino acid deletions, up to 25 amino acidinsertions, or a combination thereof.

In some embodiments, the application provides Fc fusion proteinscomprising a ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain comprises a loop,AB; a loop, BC; a loop, CD; a loop, DE; a loop, EF; and a loop, FG; andhas at least one loop selected from loop BC, DE, and FG with an alteredamino acid sequence relative to the sequence of the corresponding loopof the human ¹⁰Fn3 domain. In some embodiments, the BC and FG loops arealtered. In some embodiments, the BC, DE, and FG loops are altered,i.e., the ¹⁰Fn3 domain comprises non-naturally occurring loops. By“altered” is meant one or more amino acid sequence alterations relativeto a template sequence (i.e., the corresponding human fibronectindomain) and includes amino acid additions, deletions, and substitutions.Altering an amino acid sequence may be accomplished through intentional,blind, or spontaneous sequence variation, generally of a nucleic acidcoding sequence, and may occur by any technique, for example, PCR,error-prone PCR, or chemical DNA synthesis.

In certain embodiments, the application provides Fc fusion proteinscomprising a ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain can be definedgenerally by the following core amino acid sequence: EVVAAT(X)_(a)SLLI(X)_(x) YYRITYGE(X)_(b) QEFTV(X)_(y) ATI(X)_(c) DYTITVYAV(X)_(z)ISINYRT (SEQ ID NO:2).

In SEQ ID NO:2, the AB loop is represented by X_(a), the CD loop isrepresented by X_(b), the EF loop is represented by X_(c), the BC loopis represented by X_(x), the DE loop is represented by X_(y), and the FGloop is represented by X_(z). X represents any amino acid and thesubscript following the X represents an integer of the number of aminoacids. In particular, a may be anywhere from 1-15, 2-15, 1-10, 2-10,1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3, or 1-2 amino acids; and b, c, x,y and z may each independently be anywhere from 2-20, 2-15, 2-10, 2-8,5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 aminoacids. In preferred embodiments, a is 2 amino acids, b is 7 amino acids,c is 7 amino acids, x is 9 amino acids, y is 6 amino acids, and z is 12amino acids. The sequences of the beta strands (underlined in SEQ ID NO:2) may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,deletions or additions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 2. In an exemplaryembodiment, the sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 conservative substitutions across all 7scaffold regions relative to the corresponding amino acids shown in SEQID NO: 2. In certain embodiments, the hydrophobic core amino acidresidues are fixed and any substitutions, conservative substitutions,deletions or additions occur at residues other than the core amino acidresidues. In exemplary embodiments, the BC, DE, and FG loops asrepresented by (X)_(x), (X)_(y), and (X)_(z), respectively, are replacedwith polypeptides comprising BC, DE and FG loop sequences that bind tospecific targets.

In certain embodiments, the application provides Fc fusion proteinscomprising a ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain can be definedgenerally by the sequence: EVVAATPTSLLI(X)_(x)YYRITYGETGGNSPVQEFTV(X)_(y) ATISGLKPGVDYTITVYAV(X)_(z),IS INYRT (SEQ IDNO:3).

In SEQ ID NO:3, the BC loop is represented by X_(x), the DE loop isrepresented by X_(y), and the FG loop is represented by X_(z). Xrepresents any amino acid and the subscript following the X representsan integer of the number of amino acids. In particular, x, y and z mayeach independently be anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15,5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. Inpreferred embodiments, x is 9 amino acids, y is 6 amino acids, and z is12 amino acids. The sequences of the beta strands and south pole loops(underlined in SEQ ID NO: 3) may have anywhere from 0 to 10, from 0 to8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, orfrom 0 to 1 substitutions, deletions or additions across all 7 scaffoldregions and south pole loops relative to the corresponding amino acidsshown in SEQ ID NO: 3. In an exemplary embodiment, the sequences of thebeta strands and south pole loops may have anywhere from 0 to 10, from 0to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2,or from 0 to 1 conservative substitutions across all 7 scaffold regionsand south pole loops relative to the corresponding amino acids shown inSEQ ID NO: 3. In certain embodiments, the core amino acid residues arefixed and any substitutions, conservative substitutions, deletions oradditions occur at residues other than the core amino acid residues. Inexemplary embodiments, the BC, DE, and FG loops as represented by(X)_(x), (X)_(y), and (X)_(z), respectively, are replaced withpolypeptides comprising BC, DE and FG loop sequences that bind tospecific targets.

A ¹⁰Fn3 domain as described herein may optionally contain a modified N-and/or C-terminal sequence. For example, with reference to SEQ ID NO:2or 3, the ¹⁰Fn3 domain may comprise an N-terminal extension and/or aC-terminal tail as described further below.

In certain embodiments, the ¹⁰Fn3 domain as shown in SEQ ID NO: 2 or 3may optionally comprise an N-terminal extension of from 1-20, 1-15,1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length. ExemplaryN-terminal extensions include (represented by the single letter aminoacid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 4), VSDVPRDL (SEQ ID NO: 5),and GVSDVPRDL (SEQ ID NO: 6), or N-terminal truncations of any one ofSEQ ID NOs: 4, 5 or 6. Other suitable N-terminal extensions include, forexample, X_(n)SDVPRDL (SEQ ID NO: 7), X_(n)DVPRDL (SEQ ID NO: 8),X_(n)VPRDL (SEQ ID NO: 9), X_(n)PRDL (SEQ ID NO: 10), X_(n)RDL (SEQ IDNO: 11), X_(n)DL (SEQ ID NO: 12), or X_(n)L, wherein n=0, 1 or 2 aminoacids, wherein when n=1, X is Met or Gly, and when n=2, X is Met-Gly.When a Met-Gly sequence is added to the N-terminus of a ¹⁰Fn3 domain,the M will usually be cleaved off, leaving a G at the N-terminus.

In certain embodiments, the ¹⁰Fn3 domain as shown in SEQ ID NO: 2 or 3may optionally comprise a C-terminal tail of from 1-20, 1-15, 1-10, 1-8,1-5, or 1-4 amino acids in length. Specific examples of tail sequencesinclude, for example, polypeptides comprising, consisting essentiallyof, or consisting of, EIEK (SEQ ID NO: 13), EGSGC (SEQ ID NO: 14),EIEKPCQ (SEQ ID NO: 15), EIEKPSQ (SEQ ID NO: 16), EIEKP (SEQ ID NO: 17),EIEKPS (SEQ ID NO: 18), EIEKPC (SEQ ID NO: 19), EIDKPSQ (SEQ ID NO: 20),or EIDKPSQLE (SEQ ID NO: 21). In certain embodiments, the ¹⁰Fn3 domaincomprises a C-terminal tail comprising a sequence X(ED)_(n), wherein nis an integer from 2-10, 2-8, 2-5, 3-10, 3-8, 3-7, 3-5, 4-7, or whereinn is 2, 3, 4, 5, 6, 7, 8, 9 or 10, and X is optional, and when presentis an E, I or EI. Such ED repeat tails may enhance solubility and/orreduce aggregation of the ¹⁰Fn3 domain. In exemplary embodiments, theC-terminal tail comprises, consists essentially of, or consists of theamino acid sequence of SEQ ID NO: 15. In preferred embodiments, theC-terminal sequences lack DK sequences.

In certain embodiments, the fibronectin based scaffold proteins comprisea ¹⁰Fn3 domain having both an N-terminal extension and a C-terminaltail.

In certain embodiments, a ¹⁰Fn3 domain is a domain set forth in WO2012/016245.

Multivalent Fibronectin Based Scaffold Proteins

In certain embodiments, the application provides an Fc fusion proteincomprising a polypeptide having two or more ¹⁰Fn3 domains, e.g., amultivalent fibronectin based scaffold protein. For example, amultivalent fibronectin based scaffold protein may comprise 2, 3 or more¹⁰Fn3 domains that are covalently associated. In exemplary embodiments,the fibronectin based scaffold protein is a bispecific or dimericprotein comprising two ¹⁰Fn3 domains. In certain embodiments, amultivalent fibronectin based protein scaffold comprises a first ¹⁰Fn3domain that binds to a first target molecule and a second ¹⁰Fn3 domainthat binds to a second target molecule. The first and second targetmolecules may be the same or different target molecules. When the firstand second target molecules are the same, the ¹⁰Fn3 domains, i.e., thebinding loops, may be the same or different. Furthermore, when the firstand second ¹⁰Fn3 domains bind to the same target, they may bind to thesame or different epitopes on the target.

In exemplary embodiments, each ¹⁰Fn3 domain of a multivalent fibronectinbased protein scaffold binds to a desired target with a K_(D) of lessthan 1 mM, 100 μM, 10 μM, 1 μM, 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 100pM or less. In exemplary embodiments, each ¹⁰Fn3 domain of a multivalentfibronectin based protein scaffold binds specifically to a target thatis not bound by a wild-type ¹⁰Fn3 domain, particularly the wild-typehuman ¹⁰Fn3 domain. In exemplary embodiments, none of the ¹⁰Fn3 domainsof a multivalent fibronectin based protein scaffold bind to an integrinprotein.

In the case of multivalent fibronectin based scaffold proteins,preferably none of the ¹⁰Fn3 domains comprise a C-terminal tailcontaining a DK sequence. In exemplary embodiments, a multivalentfibronectin based scaffold protein comprises two or more ¹⁰Fn3 domains,wherein each domain comprises a C-terminal tail that does not contain aDK sequence. In certain embodiments, a multivalent fibronectin basedscaffold protein comprises two or more ¹⁰Fn3 domains, wherein eachdomain comprises a C-terminal tail that does not contain a DK sequence.

The ¹⁰Fn3 domains in a multivalent fibronectin based scaffold proteinmay be connected by a peptide linker. Exemplary peptide linkers includepeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2 aminoacids. Suitable linkers for joining the ¹⁰Fn3 domains are those whichallow the separate domains to fold independently of each other forming athree dimensional structure that permits high affinity binding to atarget molecule. In some embodiments, suitable linkers that allow theseparate domains or portions to fold independently of each othercomprise glycine-serine based linkers, glycine-proline based linkers andproline-alanine based linkers. The Examples described in WO 2009/142773demonstrate that Fn3 domains joined via these linkers retain theirtarget binding function. In some embodiments, the linker is aglycine-serine based linker. These linkers comprise glycine and serineresidues and may be between 8 and 50, 10 and 30, and 10 and 20 aminoacids in length. Examples of such linkers include GSGSGSGSGS (SEQ ID NO:32), GSGSGSGSGSGS (SEQ ID NO: 33), GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 34),GGGGSGGGGSGGGGS (SEQ ID NO: 35), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:80), and GGGGSGGGGSGGGSG (SEQ ID NO: 36). In some embodiments, thelinker is a glycine-proline based linker. These linkers comprise glycineand proline residues and may be between 3 and 30, 10 and 30, and 3 and20 amino acids in length. Examples of such linkers include GPG (SEQ IDNO: 39), GPGPGPG (SEQ ID NO: 40) and GPGPGPGPGPG (SEQ ID NO: 41). Insome embodiments, the linker is a proline-alanine based linker. Theselinkers comprise proline and alanine residues and may be between 3 and30, 10 and 30, 3 and 20 and 6 and 18 amino acids in length. Examples ofsuch linkers include PAPAPA (SEQ ID NO: 42), PAPAPAPAPAPA (SEQ ID NO:43) and PAPAPAPAPAPAPAPAPA (SEQ ID NO: 44). In other embodiments, thelinker comprises the sequence PSTSTST (SEQ ID NO: 71). It iscontemplated, that the optimal linker length and amino acid compositionmay be determined by routine experimentation based on the teachingsprovided herein. In exemplary embodiments, the linker does not containany DK sequences.

Vectors & Polynucleotides

In other embodiments, the application provides nucleic acids encodingany of the various Fc fusion proteins disclosed herein. Codon usage maybe selected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammalianceils, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21,2003); Sinclair et al., Protein Expr. Purif., 26(I):96-105 (October2002); Connell, N.D., Curr. Opin. Biotechnol., 12(5):446-449 (October2001); Makrides et al., Microbiol Rev., 60(3):512-538 (September 1996);and Sharp et al., Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), orAusubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates,herein incorporated by reference. Generally, the DNA encoding thepolypeptide is operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, viral, orinsect genes. Such regulatory elements include a transcriptionalpromoter, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites, and sequencesthat control the termination of transcription and translation. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants is additionally incorporated.

The Fc fusion proteins described herein may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. An exemplary N-terminal leader sequence forproduction of polypeptides in a mammalian system is METDTLLLWVLLLWVPGSTG(SEQ ID NO: 29), which is removed by the host cell following expression.

For prokaryotic host cells that do not recognize and process a nativesignal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces alpha-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in U.S. Pat. No. 5,631,144. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available. The DNA for suchprecursor regions may be ligated in reading frame to DNA encoding theprotein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein disclosed herein, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tan promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein disclosed herein. Promoter sequences are known foreukaryotes. Virtually all eukaryotic genes have an AT-rich regionlocated approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tall to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding proteins disclosed herein by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the peptide-encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of mRNA encoding the protein disclosed herein.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York(1985)), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney ceils, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

Protein Production

In other aspects, the application provides host cells containing vectorsencoding the Fc fusion proteins described herein, as well as methods forproducing the Fc fusion proteins described herein. Host cells may betransformed with the herein-described expression or cloning vectors forprotein production and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Host cells usefulfor high-throughput protein production (HTPP) and mid-scale productioninclude the HMS174-bacterial strain. The host cells used to produce theproteins disclosed herein may be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma)) are suitable for culturing thehost cells. In addition, may of the media described in Ham et al., Meth.Enzymol., 58:44 (1979), Barites et al., Anal. Biochem., 102:255 (1980),U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469,6,048,728, 5,672,502, or U.S. Pat. No. RE 30,985 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGentamycin drug), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

The Fc fusion proteins provided herein can also be produced usingcell-translation systems. For such purposes the nucleic acids encodingthe fusion protein must be modified to allow in vitro transcription toproduce mRNA and to allow cell-free translation of the mRNA in theparticular cell-free system being utilized (eukaryotic such as amammalian or yeast cell-free translation system or prokaryotic such as abacterial ceil-free translation system.

The Fc fusion proteins disclosed herein can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2nd Edition, The Pierce Chemical Co., Rockford, Ill. (1984)).Modifications to the Fc fusion proteins can also be produced by chemicalsynthesis.

The Fc fusion proteins disclosed herein can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, getfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrant distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified Fc fusion proteins is preferably at least 85% pure, orpreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the Fc fusionprotein is sufficiently pure for use as a pharmaceutical product.

Exemplary Uses

In one aspect, the application provides Fc fusion proteins that areuseful as diagnostic or therapeutic agents. Fc fusion proteins useful asdiagnostic agents may be labeled with a detectable moiety. The Fc fusionproteins may be used for a variety of diagnostic applications. Thedetectable moiety can be any one which is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the detectablemoiety may be a radioisotope, such as H3, C14, C13, P32, S35, or I131; afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for conjugating a protein to the detectablemoiety may be employed, including those methods described by Hunter, etal., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974);Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982). In vitro methods, include conjugationchemistry well know in the art including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a detectable moiety to an Fc protein, a linkinggroup or reactive group is used. Suitable linking groups are well knownin the art and include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups and esterase labilegroups. Preferred linking groups are disulfide groups and thioethergroups depending on the application. For polypeptides without a Cysamino acid, a Cys can be engineered in a location to allow for activityof the protein to exist while creating a location for conjugation.

Fc fusion proteins linked with a detectable moiety are useful for invitro or in vivo imaging. The polypeptide may be linked to aradio-opaque agent or radioisotope, administered to a subject,preferably into the bloodstream, and the presence and location of thelabeled protein in the subject may be assayed. This imaging technique isuseful, for example, in the staging and treatment of malignancies whenthe Fc fusion protein binds to a target associated with cancer. The Fcfusion protein may be labeled with any moiety that is detectable in asubject, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

Fc fusion proteins also are useful as affinity purification agents. Inthis process, the Fc fusion proteins are immobilized on a suitablesupport, such as Sephadex resin or filter paper, using methods wellknown in the art.

Fc fusion proteins can be employed in any known assay method, such ascompetitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987)).

In certain aspects, the disclosure provides methods for detecting atarget molecule in a sample. A method may comprise contacting the samplewith an Fc fusion protein described herein, wherein said contacting iscarried out under conditions that allow the Fc fusion protein-targetcomplex formation; and detecting said complex, thereby detecting saidtarget in said sample. Detection may be carried out using any techniqueknown in the art, such as, for example, radiography, immunologicalassay, fluorescence detection, mass spectroscopy, or surface plasmonresonance. The sample will often by a biological sample, such as abiopsy, and particularly a biopsy of a tumor, or a suspected tumor,where the Fc fusion protein binds to a target associated with cancer.The sample may be from a human or other mammal. The Fc fusion proteinmay be labeled with a labeling moiety, such as a radioactive moiety, afluorescent moiety, a chromogenic moiety, a chemiluminescent moiety, ora hapten moiety. The Fc fusion protein may be immobilized on a solidsupport.

In one aspect, the application provides Fc fusion proteins useful in thetreatment of disorders. The diseases or disorders that may be treatedwill be dictated by the identity of the protein fused to the Fc domain.Exemplary therapeutic proteins that may be bound to an Fc domaininclude, for example, interferon alpha (for treating hepatitis),L-asparaginase (for the treatment of acute lymphoblastic leukemia), orgranulocyte colony-stimulating factor (for treatment of cancerchemotherapy induced neutropenia). In certain embodiments, the Fc fusionproteins described herein comprise an antibody, or fragment thereof,such as, for example, and anti-TNF-alpha antibody (for the treatment ofautoimmune diseases like rheumatoid arthritis or Crohn's disease). In anexemplary embodiment, the Fc fusion protein described herein comprise apolypeptide comprising ¹⁰Fn3 domain, including, for example, apolypeptide comprising a ¹⁰Fn3 domain that binds to a target such astumor necrosis factor alpha (TNF-alpha), delta-like protein 4 (DLL4),interleukin 17 (IL-17), proprotein convertase subtilisin kexin type 9(PCSK9), pregnane X receptor (PXR), epidermal growth factor receptor(EGFR), insulin-like growth factor 1 receptor (IGF-1R), vascularendothelial growth factor receptor (VEGFR2), and interleukin 23 (IL-23).¹⁰Fn3 domains that bind to TNF-alpha may be used to treat autoimmunedisorders such as rheumatoid arthritis, inflammatory bowel disease,psoriasis, and asthma; ¹⁰Fn3 domains that bind to IL-17 may be used totreat asthma; ¹⁰Fn3 domains that bind to DLL4, EGFR, VEGFR2 or IGF-1Rmay be used to treat hyperproliferative disorders or diseases associatedwith unwanted angiogenesis, such as cancers or tumors; and ¹⁰Fn3 domainsthat bind to PCSK9 may be used to treat atherosclerosis,hypercholesterolemia and other cholesterol related diseases.

The application also provides methods for administering Fc fusionproteins to a subject. In some embodiments, the subject is a human. Insome embodiments, the Fc fusion proteins are pharmaceutically acceptableto a mammal, in particular a human. A “pharmaceutically acceptable”composition refers to a composition that is administered to an animalwithout significant adverse medical consequences. Examples ofpharmaceutically acceptable compositions include compositions that areessentially endotoxin or pyrogen free or have very low endotoxin orpyrogen levels.

Formulation and Administration

The application further provides pharmaceutically acceptablecompositions comprising the Fc fusion proteins described herein.Therapeutic formulations comprising Fc fusion proteins are prepared forstorage by mixing the described proteins having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The Fc fusion proteins may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the fibronectin based scaffold proteinsdescribed herein, which matrices are in the form of shaped articles,e.g., films, or microcapsule. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated proteins remain inthe body for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

While the skilled artisan will understand that the dosage of each Fcfusion protein will be dependent on the identity of the protein, thepreferred dosages can range from about 10 mg/square meter to about 2000mg/square meter, more preferably from about 50 mg/square meter to about1000 mg/square meter.

For therapeutic applications, the Fc fusion proteins are administered toa subject, in a pharmaceutically acceptable dosage form. They can beadministered intravenously as a bolus or by continuous infusion over aperiod of time, by intramuscular, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. Theprotein may also be administered by intratumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects. Suitable pharmaceutically acceptablecarriers, diluents, and excipients are well known and can be determinedby those of skill in the art as the clinical situation warrants.Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH about 7.4, containing about 1mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl),and (3) 5% (w/v) dextrose. The methods of the present invention can bepracticed in vitro, in vivo, or ex vivo.

Administration of Fc fusion proteins, and one or more additionaltherapeutic agents, whether co-administered or administeredsequentially, may occur as described above for therapeutic applications.Suitable pharmaceutically acceptable carriers, diluents, and excipientsfor co-administration will be understood by the skilled artisan todepend on the identity of the particular therapeutic agent beingco-administered.

When present in an aqueous dosage form, rather than being lyophilized,the Fc fusion protein typically will be formulated at a concentration ofabout 0.1 mg/ml to 100 mg/ml, although wide variation outside of theseranges is permitted. For the treatment of disease, the appropriatedosage of Fc fusion proteins will depend on the type of disease to betreated, the severity and course of the disease, whether the Fc fusionproteins are administered for preventive or therapeutic purposes, thecourse of previous therapy, the patient's clinical history and responseto the Fc fusion protein, and the discretion of the attending physician.The Fc fusion protein is suitably administered to the patient at onetime or over a series of treatments.

Sequence listing WT ¹⁰Fn3 SequenceVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 1)WT Core ¹⁰Fn3 Sequence EVVAAT(X)_(a) SLLI(X)_(x) YYRITYGE(X)_(b)QEFTV(X)_(y) ATI(X)_(c) DYTITVYAV(X)_(z) ISINYRT (SEQ ID NO: 2)EVVAATPTSLLI(X)_(x)YYRITYGETGGNSPVQEFTV(X)_(y)ATISGLKPGVDYTITVYAV(X)_(z) IS INYRT (SEQ ID NO: 3)MGVSDVPRDL (SEQ ID NO: 4) VSDVPRDL (SEQ ID NO: 5)GVSDVPRDL (SEQ ID NO: 6) X_(n)SDVPRDL (SEQ ID NO: 7)X_(n)DVPRDL (SEQ ID NO: 8) X_(n)VPRDL (SEQ ID NO: 9)X_(n)PRDL (SEQ ID NO: 10) X_(n)RDL (SEQ ID NO: 11)X_(n)DL (SEQ ID NO: 12) EIEK (SEQ ID NO: 13) EGSGC (SEQ ID NO: 14)EIEKPCQ (SEQ ID NO: 15) EIEKPSQ (SEQ ID NO: 16) EIEKP (SEQ ID NO: 17)EIEKPS (SEQ ID NO: 18) EIEKPC (SEQ ID NO: 19) EIDKPSQ (SEQ ID NO: 20)EIDKPSQLE (SEQ ID NO: 21) Human IgG1 Constant RegionASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22)DKTHTCPPCPAPELLG (SEQ ID NO: 23) EPKSSDKTHTCPPCPAPELLGGPS(SEQ ID NO: 24; core hinge region underlined) EPKSSDKTHTCPPCPAPELLGGSS(SEQ ID NO: 25; core hinge region underlined) EPKSSGSTHTCPPCPAPELLGGSS(SEQ ID NO: 26; core hinge region underlined) DKTHTCPPCPAPELLGGPS(SEQ ID NO: 27; core hinge region underlined) DKTHTCPPCPAPELLGGSS(SEQ ID NO: 28, core hinge region underlined)METDTLLLWVLLLWVPGSTG (SEQ ID NO: 29) PRD460 Amino Acid SequenceGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTEI EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30)CH2 and CH3 Regions of Human IgG1VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31)GSGSGSGSGS (SEQ ID NO: 32) GSGSGSGSGSGS (SEQ ID NO: 33)GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 34) GGGGSGGGGSGGGGS (SEQ ID NO: 35)GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 80)GGGGSGGGGSGGGSG (SEQ ID NO: 36) AGGGGSG (SEQ ID NO: 37)AGGGGSGG (SEQ ID NO: 38) GPG (SEQ ID NO: 39) GPGPGPG (SEQ ID NO: 40)GPGPGPGPGPG (SEQ ID NO: 41) PAPAPA (SEQ ID NO: 42)PAPAPAPAPAPA (SEQ ID NO: 43) PAPAPAPAPAPAPAPAPA (SEQ ID NO: 44)QPDEPGGS (SEQ ID NO: 45) ELQLEESAAEAQDGELD (SEQ ID NO: 46)TVAAPS (SEQ ID NO: 47) QPDEPGGSG (SEQ ID NO: 48)ELQLEESAAEAQDGELDG (SEQ ID NO: 49) TVAAPSG (SEQ ID NO: 50)SCSVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 51)SCCVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 52)DWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 53)SCCVADWQMPPPYVVLDLPQETLEEETPGAN (SEQ ID NO: 54)YLAMTPLIPQSKDENSDDYTTFDDVGS (SEQ ID NO: 55)ELDVCVEEAEGEAPW (SEQ ID NO: 56) ELQLEESCAEAQDGELDG (SEQ ID NO: 57)EGEVSADEEGFEN (SEQ ID NO: 58) KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 59)KPTHVNVSVVMAEVDGTCY (SEQ ID NO: 60) YVTDHGPMK (SEQ ID NO: 61)PTLYNVSLVMSDTAGTCY (SEQ ID NO: 62) SXSVADWQMPPPYVVLDLPQETLEEETPGAN,wherein X is serine, alanine or glycine (SEQ ID NO: 63)SXXVADWQMPPPYVVLDLPQETLEEETPGAN,wherein each X is independently selectedfrom serine, alanine or glycine (SEQ ID NO: 64)SXXVADWQMPPPYVVLDLPQETLEEETPGAN,wherein each X is independently selectedfrom serine, alanine or glycine (SEQ ID NO: 65) ELDVXVEEAEGEAPW, wherein X is serine, alanine or glycine (SEQ ID NO: 66)ELQLEESXAEAQDGELDG, wherein X is serine, alanine or glycine(SEQ ID NO: 67) KPTHVNVSVVMAEVDGTXY,wherein X is serine, alanine or glycine (SEQ ID NO: 68)KPTHVNVSVVMAEVDGTXY, wherein X is serine, alanine or glycine(SEQ ID NO: 69) PTLYNVSLVMSDTAGTXY, wherein X is serine, alanine or glycine (SEQ ID NO: 70)PSTSTST (SEQ ID NO: 71) ATI-1174 Amino Acid SequenceMGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTEIEKPCQ (SEQ ID NO: 72)ATI-1174 Nucleic Acid SequenceATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGAGAAACCATGCCAGTG (SEQ ID NO: 73)ATI-1081 Amino Acid SequenceMGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTEIDKPSQ (SEQ ID NO: 74)ATI-1081 Nucleic Acid SequenceATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 75)ATI-1114 Amino Acid SequenceMGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTGSGC (SEQ ID NO: 76)ATI-1114 Nucleic Acid SequenceATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 77)ATI-972 Amino Acid SequenceMGVSDVPRDLEVVAATPTSLLISWPPPSHGYGYYRITYGETGGNSPVQEFTVPPGKGTATISGLKPGVDYTITVYAVEYPYKHSGYYHRPISINYRTEIDKPCQ (SEQ ID NO: 78)ATI-972 Nucleic Acid SequenceATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGCCGCCGCCGTCTCATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCGCCTGGTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACAAACATTCTGGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATGCCAGCACCATCACCACCACCAC (SEQ ID NO: 79)QPDEP (SEQ ID NO: 81) PVPPPPP (SEQ ID NO: 82)EDEDEDEDEDE (SEQ ID NO: 83) DLPQETLEEETPGA (SEQ ID NO: 84)VPSTPPTPSPST (SEQ ID NO: 85) ELQLEESAAEAQEGELE (SEQ ID NO: 86)ESPKAQASSVPTAQPQAE (SEQ ID NO: 87) PAVPPP (SEQ ID NO: 88)EPKSSDKTHTCPPCP (SEQ ID NO: 89) VPSTPPTPSPSTG (SEQ ID NO: 90)VPSTPPTPSPSTPPTPSPSG (SEQ ID NO: 91) GRGGEEKKKEKEKEEG (SEQ ID NO: 92)GRGGEEKKKEKEKEEQEERETKTPG (SEQ ID NO: 93) ESPKAQASSG (SEQ ID NO: 94)ESPKAQASSVPTAQPQAEG (SEQ ID NO: 95)SVEEKKKEKEKEEQEERETKTPG (SEQ ID NO: 96)PSVEEKKKEKEKEEQEERETKTPG (SEQ ID NO: 97)GSVEEKKKEKEKEEQEERETKTPG (SEQ ID NO: 98) Fc4EPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 99)Fc5 EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 100) Fc6EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 101)Fc7 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 102) Fc8EPRSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 103) Fc9DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 104)Fc10 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 105) Fc11EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 106) Fc12EPKSSDKTHTSPPSPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 107)Fc13 EPKSSDKTHTSPPSPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108)Fc14 EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109)Fc15 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 110)Fc16 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 111) Fc17EPKSSDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 112) Fc18EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK(SEQ ID NO: 113) Fc19EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 114) Fc21EPKSSDKTHTSPPSPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 115)Fc22 EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116)Fc23 EPKSSDKTHTCPPCPAPEAGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 117) mFc1EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 118) mFc3EPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK (SEQ ID NO: 119) mFc2EPRSPTIKPCPPCKCPAPNLEGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKAFACAVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 120) mFc4EPRSPITQNPCPPLKECPPCAAPDLEGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKAFACAVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK (SEQ ID NO: 121) PRD289GVSDVPRDLEVVAATPTSLLISWRPPIHAYGYYRITYGETGGNSPVQEFTVPIVEGTATISGLKPGVDYTITVYAVEYTFKHSGYYHRPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 122) PRD292EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGVSDVPRDLEVVAATPTSLLISWRPPIHAYGYYRITYGETGGNSPVQEFTVPIVEGTATISGLKPGVDYTITVYAVEYTFKHSGYYHRPISINYRTEI (SEQ ID NO: 123) PRD290GVSDVPRDLEVVAATPTSLLISWSPPANGYGYYRITYGETGGNSPVQEFTVPVGRGTATISGLKPGVDYTITVYAVEYTYKGSGYYHRPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 124) PRD293EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGVSDVPRDLEVVAATPTSLLISWSPPANGYGYYRITYGETGGNSPVQEFTVPVGRGTATISGLKPGVDYTITVYAVEYTYKGSGYYHRPISINYRTEI (SEQ ID NO: 125) PRD713GVSDVPRDLEVVAATPTSLLISWGHYPLHVRYYRITYGETGGNSPVQEFTVPPRSHTATISGLKPGVDYTITVYAVTYYAQENYKEIPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 126) PRD239EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGVSDVPRDLEVVAATPTSLLISWGHYPLHVRYYRITYGETGGNSPVQEFTVPPRSHTATISGLKPGVDYTITVYAVTYYAQENYKEIPISINYRTEAS (SEQ ID NO: 127)C7FL-Fc (PRD1309)GSVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 128) C7FL-Fc (PRD1308)GSVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 129)

EXAMPLES

The invention now being generally described will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Anti-PCSK9 Adnectin Clones

¹⁰Fn3 domains that bound with affinity to PCSK9 were identified usingthe ProFusion method. See e.g., WO02/032925.

ATI-1174 is a pegylated anti-PCSK9 Adnectin having the following aminoacid sequence:MGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTEIEKPCQ (SEQ ID NO: 72).

ATI-1174 is encoded by the following nucleotide sequence:

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGAGAAACCATGCCAGTG (SEQ ID NO: 73).

ATI-1081 is an anti-PCSK9 Adnectin having the following amino acidsequence and a 6×His tag:

MGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTEIDKPSQ (SEQ ID NO: 74).

ATI-1081 is encoded by the following nucleotide sequence:

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO: 75).

ATI-1114 is a pegylated anti-PCSK9 adnectin that is a derivative ofATI-1081 having a different C-terminal tail sequence and a 6×His tag:

MGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTGSGC (SEQ ID NO: 76).

ATI-1114 is encoded by the following nucleotide sequence:

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO: 77).

ATI-972 is a biotinylated anti-PCSK9 adnectin with 6-histidinec-terminus and biotinylation at cysteine, and having the followingsequence:

MGVSDVPRDLEVVAATPTSLLISWPPPSHGYGYYRITYGETGGNSPVQEFTVPPGKGTATISGLKPGVDYTITVYAVEYPYKHSGYYHRPISINYRTEIDKPCQ (SEQ ID NO: 78).

ATI-972 is encoded by the following nucleotide sequence:

ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGCCGCCGCCGTCTCATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCGCCTGGTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACAAACATTCTGGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATGCCAGCACCATCACCACCACCAC (SEQ ID NO: 79).

PRD460 is an anti-PCSK9 Adnectin-Fc fusion proteins having the followingamino acid sequence:GVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30). The ¹⁰Fn3 domain thatbinds PCSK9 is shown in italics; the hinge sequence is underlined; andthe CH2 and CH3 regions shown in regular text are derived from IgG1.

The anti-PCSK9 adnectins may be expressed in E. coli with an N-terminalmethionine, or in mammalian cells with the following leader sequence:METDTLLLWVLLLWVPGSTG (SEQ ID NO: 29).

Example 2 Protein Production and Purification

Midscale Expression and Purification of Insoluble Fibronectin-BasedScaffold Protein Binders

For expression of insoluble clones, the clone(s), followed by theHIS₆tag, are cloned into a pET9d (EMD Bioscience, San Diego, Calif.)vector and are expressed in E. coli HMS174 cells. Twenty ml of aninoculum culture (generated from a single plated colony) is used toinoculate 1 liter of LB medium containing 50 μg/ml carbenicillin and 34μg/ml chloromphenicol. The culture is grown at 37° C. until A₆₀₀0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG)the culture is grown for 4 hours at 30° C. and is harvested bycentrifugation for 30 minutes at ≧10,000 g at 4° C. Cell pellets arefrozen at −80° C. The cell pellet is resuspended in 25 ml of lysisbuffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) using an Ultra-turraxhomogenizer (IKA works) on ice. Cell lysis is achieved by high pressurehomogenization (≧18,000 psi) using a Model M-110S MICROFLUIDIZER®(Microfluidics). The insoluble fraction is separated by centrifugationfor 30 minutes at 23,300 g at 4° C. The insoluble pellet recovered fromcentrifugation of the lysate is washed with 20 mM sodiumphosphate/500 mMNaCl, pH7.4. The pellet is resolubilized in 6.0M guanidine hydrochloridein 20 mM sodium phosphate/500M NaCl pH 7.4 with sonication followed byincubation at 37 degrees for 1-2 hours. The resolubilized pellet isfiltered to 0.45 μm and loaded onto a Histrap column equilibrated withthe 20 mM sodium phosphate/500M NaCl/6.0M guanidine pH 7.4 buffer. Afterloading, the column is washed for an additional 25 CV with the samebuffer. Bound protein is eluted with 50 mM Imidazole in 20 mM sodiumphosphate/500 mM NaCl/6.0M guan-HCl pH7.4. The purified protein isrefolded by dialysis against 50 mM sodium acetate/150 mM NaCl pH 4.5.

Midscale Expression and Purification of Soluble Fibronectin-BaseScaffold Protein Binders

For expression of soluble clones, the clone(s), followed by the HIS₆tag,were cloned into a pET9d (EMD Bioscience, San Diego, Calif.) vector andwere expressed in E. coli HMS174 cells. Twenty ml of an inoculum culture(generated from a single plated colony) was used to inoculate 1 liter ofLB medium containing 50 μg/ml carbenicillin and 34 μg/mlchloromphenicol. The culture was grown at 37° C. until A₆₀₀ 0.6-1.0.After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), theculture was grown for 4 hours at 30° C. and was harvested bycentrifugation for 30 minutes at ≧10,000 g at 4° C. Cell pellets werefrozen at −80° C. The cell pellet was resuspended in 25 ml of lysisbuffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) using an Ultra-turraxhomogenizer (IKA works) on ice. Cell lysis was achieved by high pressurehomogenization (18,000 psi) using a Model M-110S MICROFLUIDIZER®(Microfluidics). The soluble fraction was separated by centrifugationfor 30 minutes at 23,300 g at 4° C. The supernatant was clarified via0.45 μm filter. The clarified lysate was loaded onto a Histrap column(GE) pre-equilibrated with the 20 mM sodium phosphate/500M NaCl pH 7.4.The column was then washed with 25 column volumes of the same buffer,followed by 20 column volumes of 20 mM sodium phosphate/500M NaCl/25 mMImidazole, pH 7.4 and then 35 column volumes of 20 mM sodiumphosphate/500M NaCl/40 mM Imidazole, pH 7.4. Protein was eluted with 15column volumes of 20 mM sodium phosphate/500M NaCl/500 mM Imidazole, pH7.4, fractions were pooled based on absorbance at A₂₈₀ and were dialyzedagainst 1×PBS, 50 mM Tris, 150 mM NaCl. pH 8.5 or 50 mM NaOAc; 150 mMNaCl; pH4.5. Any precipitate was removed by filtering at 0.22 μm.

Fc fusions can be made in mammalian cells or in E. coli.

Example 3 PRD460 K_(D) by SPR

A vector encoding PRD460 was transfected into HEK-293 6E cells usingpolyethylenimine (PEI). The cells were grown at 37° C. for 5 days with80% humidification and 5% CO₂. The cells were then pelleted, thesupernatant was passed through a 0.22 um filter and then loaded onto toa ProteinA column. The column was washed with PBS and the protein waseluted with 20 mM Gylcine, 150 mM NaCl pH 2.8. The eluted protein wasconcentrated and passed over a superdex200 column in 50 mM MES, 100 mMNaCl pH 5.8.

The binding characteristics were characterized by Surface PlasmonResonance (SPR). Anti-human antibody was immobilized on a Biacore chip,and PRD460 was captured on the chip surface. Varying concentrations ofhPCSK9 were placed into the flow solution using MgCl2 (3 M) for chipregeneration between cycles. For comparison, ATI-1081 was captured on ananti-H is antibody immobilized on a Biacore chip. Duplicate experimentsfor PRD460 were performed on different days. Kinetic determinations wereperformed at 25° C. Evaluation of the kinetic parameters was performedusing the 1:1 Binding algorithm on the Biacore Evaluation software.

Under these conditions, ATI-1081 bound to human PCSK9 with adissociation constant (K_(D)) of 6.7 nM at 25° C. and PRD460 bound tohuman PCSK9 with a dissociation constant (K_(D)) of 3.29+/−0.55 nM at25° C., indicating equivalent binding affinity of the Fc and non-Fcformatted versions of ATI1081 (Table 1). The off-rate determinationsusing this assay format may be artificially limited by the off-rate ofthe captured ligand from the immobilized capture antibody, thus theassay format using direct immobilization of PCSK9 is a more accuratereflection of dissociation constant (K_(D)) for ATI-1081.

TABLE 1 Kinetic parameters for PRD460 and ATI-1081 against capturedhuman PCSK9 ka (1/Ms) kd (1/s) KD (nM) PRD460 3.75 +/− 0.7E+04 1.21 +/−0.05E−04 3.29 +/− 0.55 ATI-1081 3.65E+04 2.45E−04 6.7

Example 4 PCSK9 Binding FRET Assays

Two fluorescence resonance energy transfer (FRET) based assays were usedto determine the competitive binding potency of PRD460 and otheradnectins to hPCSK9. The PCSK9:EGFA FRET assay measures the binding ofPCSK9 to the LDLR, using a soluble epidermal growth factor precursorhomology domain-A (EGFA) peptide and recombinant human PCSK9. ThePCSK9:ATI972 FRET assay measures competitive displacement by adnectinsof the biotinylated adnectin, ATI-972, from PCSK9.

In the PCSK9:EGFA FRET assay (at 5 nM PCSK9), PRD460 completely andpotently displaced EGFA from the PCSK9 binding site with EC50=0.7 nM(FIG. 1, left panel). PRD460 was more potent in this assay than eitherATI-1174 (EC50=1.9 nM) or ATI-1081 (EC50=3.7 nM) (FIG. 1). The greaterapparent potency of PRD460 in this assay may be explained by bivalent(2:1) binding of adnectin PRD460 to PCSK9 (theoretically) compared tomonovalent (1:1) binding by ATI-1081 and ATI-1174.

Using the PCSK9:ATI-972 FRET assay (at 5 nM human PCSK9), PRD460inhibited with EC50=0.3 nM, compared to 0.8 nM for ATI-1114 and 2.8 nmfor ATI-1081 (FIG. 2). These findings indicate that PRD460 potentlydisplaced the biotinylated adnectin ATI-972 from its binding site onPCSK9. The higher potency of PRD460 relative to ATI-1081 and ATI-1174 isconsistent with bivalent binding by PRD460.

Example 5 Inhibition of PCSK9-Induced LDLR Depletion in HepG2 Cells

Human PCSK9 promotes the depletion of LDLR from the surface of HepG2cells. Pre-incubation of PCSK9 with PCSK9 adnectins inhibits PCSK9binding to LDLR and prevents the depletion of LDLR from the cellsurface. This assay was used to measure the potency of ATI-1081,ATI-1174 and PRD460 to inhibit PCSK9 induced depletion of LDLR from thecell surface.

A dilution series of PCSK9 adnectins were pre-incubated with 10 nM humanPCSK9 for 1 hr at 37 degrees, the pre-incubated mixture was added toHepG2 cells, and the cells were incubated for 24 hours. Following thisincubation, the level of LDLR on HepG2 cells was measured using FACSanalysis. The percentage of inhibition of PCSK9-induced LDLR depletionwas calculated and graphed (FIG. 2). In this assay ATI-1081, ATI-1174,and PRD460 inhibited PCSK9 with comparable EC50's (9 nM, 8 nM and 6 nMrespectively) although a leftward-shift of the response curve wasconsistently observed for PRD460. These EC50's represent the limit ofthe assay.

This assay was also used to determine the importance of Fc orientationon the biological activity of Fc-¹⁰Fn3 fusion proteins. To this end, theability of 1784F03 (no Fc), 1784F03-Fc (X-Fc orientation, wherein X isthe ¹⁰Fn3 domain) and Fc-1784F03 (Fc-X orientation) to inhibit PCSK9induced depletion of LDLR from the cell surface was assessed. Theability of 1813E02 (no Fc), 1813E02-Fc (X-Fc orientation) and Fc-1813E02(Fc-X orientation) to inhibit PCSK9 induced depletion of LDLR from thecell surface was also assessed.

A dilution series was prepared and pre-incubated as above with 10 nMhuman PCSK9 for 1 hr at 37 degrees, then added to HepG2 cells, and thecells were incubated for 24 hours. Following this incubation, the levelof LDLR on HepG2 cells was measured using FACS analysis. The percentageof inhibition of PCSK9-induced LDLR depletion was calculated and graphed(FIGS. 16-17, and Tables 17-18). In this assay, 1784F03, 1784F03-Fc,1813E02 and 1813E02-Fc inhibited PCSK9 with comparable IC50's (13 nM, 9nM, 10 nM and 4 nM, respectively), whereas Fc-1784F03 and Fc-1813E02 hadsignificantly higher IC50's (47 nM and 37 nM, respectively). Therefore,these results indicate that the X-Fc orientation may be important forPCSK9 ¹⁰Fn3 domains to retain their biological activity when fused to anFc moiety.

TABLE 17 Summary of HepG2 depletion inhibition by 1784F03, 1784F03-Fcand Fc- 1784F03 1784F03-Fc Fc-1784F03 1784F03 (PRD 289) (PRD 292) IC5013.24 9.150 47.77 R² 0.9934 0.9871 0.9879

TABLE 18 Summary of HepG2 depletion inhibition by 1813E02, 1813E02-Fcand Fc- 1813E02 1813E02-Fc Fc-1813E02 1813-E02 PRD 290 PRD 293 IC5010.55 4.201 37.78 R² 0.9961 0.9871 0.9745

Example 6 PCSK9 Cell Entry Assay in HepG2 Cells

PCSK9 binding to the LDLR on the surface of hepatocytes results inco-internalization of the LDLR-PCSK9 complex during LDLR endocytosis,leading to enhanced degradation of the LDLR. A cell-based assay wasdeveloped to measure LDLR-dependent cellular entry of fluorescent PCSK9.Human PCSK9 was covalently labeled using the fluorophore AlexaFluor-647(AF647). PCSK9-AF647 was incubated with HepG2 cells with orwithout PCSK9-adnectins and the intracellular fluorescence wasquantified by high content fluorescent microscopy and image analysis(Cellomics). Dependence of PCSK9-AF647 cell entry on LDLR endocytosiswas established in preliminary experiments. HepG2 cells were incubatedwith 10 nM PCSK9-AF647 and varying levels of adnectins for 4 hrs at 37degrees. In this assay, potent inhibition of PCSK9-AF647 intracellularfluorescence was observed for PRD460 (EC50=6 nM) as well as for ATI-1174(EC50=10 nM) (FIG. 3). These findings indicate that adnectin PRD460 andATI-1174 effectively and equivalently blocked the binding of PCSK9 tocell surface LDLR in a human hepatic-derived cell line in culture,thereby reducing the internalization of PCSK9-AF647 during LDLRendocytosis.

Example 7 In Vivo Transgenic Mouse Study

In vivo studies were conducted in the line 66 genomic hPCSK9 transgenicmouse model developed at BMS. This line expresses physiological levelsof hPCSK9 (˜1-5 nM). Binding of adnectins to PCSK9 in the plasma ispredicted to result in a decrease in the measured amount of unbound(free) circulating PCSK9. The decrease in unbound PCSK9 is the initialpharmacodynamic event which results in inhibition of the PCSK9-LDLRinteraction and in LDL cholesterol lowering. Administration of singledoses of PRD460 (i.p. doses from 0.6 to 18 mg/kg) to the transgenic miceresulted in rapid, strong decreases in plasma unbound hPCSK9 levels(FIG. 4). Dose-dependent decreases in unbound PCSK9 were observed withED50<0.6 mg/kg at the 3 hr time point. These findings in the normalexpresser human PCSK9 transgenic mouse model show that PRD460 bindsstrongly and potently to circulating hPCSK9 in vivo.

Example 8 In Vivo Pharmacodynamics in Cynomolgus Monkeys

The pharmacodynamic effects of PCSK9 adnectin PRD460 were evaluated innormal lean cynomolgus monkeys. PRD460 was administered to monkeys byi.v. dosing at 15 mg/kg, and plasma samples were collected at timeintervals over 4 wks for the assay of LDL-C and free PCSK9 levels. Asingle dose of PRD460 rapidly lowered plasma LDL-C levels in themonkeys, reaching an average maximum effect of 42% of baseline LDL-C(58% reduction; n=3 monkeys) by day 3 after dosing (FIG. 5). LDL-Clevels were reduced by 50% or more for a week at this dose, remainingsignificantly below baseline for 3 wks and returning to baseline by 4wks. Total cholesterol showed a similar pattern but no effect on HDL wasobserved (not shown). Treatment with PRD460 caused an immediate drop tonear zero (below the lower limit of quantitation) in the unbound, freeform of plasma PCSK9 (FIG. 5). The free PCSK9 levels remained near thelower limits of detection for several days then gradually returned tobaseline levels by the end of 4 wks, consistent with a cause/effectrelationship with plasma LDL-C. The data indicate that plasma LDLlowering mirrored the drop in free PCSK9 levels, consistent with PCSK9inhibition regulating LDLR function following treatment with PRD460 invivo. Pharmacokinetic analysis revealed that the plasma half-life ofadnectin PRD460 was approximately 70 hrs in this cynomolgus monkeystudy. These findings indicate that a PCSK9 adnectin-Fc fusion proteinis highly efficacious and fast-acting with robust, specific, andlong-lasting effects on LDL-C lowering in the cynomolgus monkey model.

Example 9 Pharmacokinetic Properties of Fc-¹⁰Fn3 Fusion Proteins

Pharmacokinetic properties of Fc-¹⁰Fn3 fusion proteins were evaluated inmice and cynomolgus monkeys. The results of these experiments aresummarized in Table 2.

TABLE 2 Summary of Pharmacokinetics properties of various ¹⁰Fn3-Fcfusion to several different proteins in mice and cynomolgus monkeys IDmouse t_(1/2) (hours) cyno t_(1/2) (hours) PRD460 96 74-78 PRD461 67 ndPRD239 61 nd PRD713 66 nd Adn-1 68 (IV) 188 (IV) 57 (SC) 335 (SC)* Adn-430 (IV) ND 25 (SC) ND Adn-5 65 (IV) ND 65 (SC) ND Adn-8 64 ND Adn-2 ND51-67 Adn-3 73 84-90 Adn-9 28-30 ND Adn-6 83 ND Adn-7 126  ND C7FLFc 2347 *t_(1/2) could not accurately be determined.Monkey In Vivo Study Designs

To determine the PK of various Fc-¹⁰Fn3 fusion proteins in monkeys,monkeys were dosed from 0.5-15 mg/kg either IV or SC with the fusionprotein of interest and serum or plasma samples were collected atspecific time points over the course of 4 weeks. Samples were collectedand processed in K₂EDTA or SST for plasma or serum, respectively, andstored at −80° C. until analysis.

ELISA/ECLA Method

In most instances, ELISA or ECLA assays were developed to determine theplasma concentration of Fc-¹⁰Fn3 fusions in mouse or monkey plasma. Ingeneral, either biotinylated target, target-Fc fusion, or anti-idiotypicantibodies were used to capture the Fc-¹⁰Fn3 fusions in plasma or serum.Detection was achieved via either an anti-hu-Fc antibody coupled to HRPor sulfo-tag, or antibodies that is binds the constant regions of the¹⁰Fn3 domain in combination with anti-rabbit-HRP or sulfo-taggedpolyclonal antibodies. In one instance, both capture and detection wereachieved via anti-hu-Fc polyclonals in which the detection antibody wascoupled to HRP. The read-out was either colorimetric via TMB orelectrochemiluminescent using the Mesoscale Discovery platform. Plasmaconcentrations were typically calculated based on a 4 or 5-parameter fitof an 8-point standard curve.

LC/MS/MS Method

In some instances, LC/MS/MS methods were developed to determine theplasma concentration of Fc-¹⁰Fn3 fusions in mouse or monkey plasma orserum. The analysis utilizes trypsin digestion of the target proteins togenerate a surrogate peptide from the Adnectin portion of the moleculesand a surrogate peptide from the Fc region. The surrogate peptides weredetected by tandem mass spectrometry. The basis of quantification is thestoichiometric relationship between Adnectin proteins and thesurrogates.

Standard curves were prepared in the same matrix as the study samples.The standard curves and study samples were subjected to thermaldenaturation followed by tryptic digestion prior to proteinprecipitation, followed by LC-MS/MS analysis. Plasma concentrations weretypically calculated based on quadratic fit of a standard curve.

Pharmacokinetic Analysis

Pharmacokinetic (PK) parameters for Fc-¹⁰Fn3 fusions were calculatedusing Phoenix WinNonlin version 6.2 (Pharsight Corp, Mountain View,Calif.) non-compartmental analysis or comparable software. The peakconcentration (Cmax) was recorded directly from experimentalobservations. The area under the curve (AUC) values were calculatedusing a combination of linear and log trapezoidal summations. The totalplasma clearance (CL_F_obs), volume of distribution (Vz_F_obs or Vss),terminal half-life (T-HALF) and mean residence time (MRT) wereestimated.

Pharmacokinetic Properties of Fc-¹⁰Fn3 Fusion Proteins in CynomolgusMonkeys.

The half-life (t_(1/2)) of PCSK9 Adnectin PRD460 (Fc-¹⁰Fn3) and that ofPCSK9 Adnectin ATI-1081 (no Fc) was determined following administrationinto cynomolgus monkeys. Results show that Fc moiety enhances thehalf-life of ¹⁰Fn3 proteins (FIG. 6 and Tables 2 and 3).

TABLE 3 Pharmacokinetic properties of PRD460 vs. ATI-1081 T-HALF V_(D)CL AUCall MRT Format (h) (mL/kg) (mL/h/kg) (h * μmol/L) (h) ATI-10811.27 385 214 4.32 1.31 PRD460 78 104 0.92 230 74

An experiment was performed to compare the half-life (t_(1/2)) ofFc-¹⁰Fn3 fusion proteins targeting soluble ligands. The pharmacokineticsof PCSK9 PRD460 and another Fc-¹⁰Fn3 fusion protein to a differentsoluble ligand target (Adn-1) were evaluated following IV administrationinto cynomolgus monkeys. Adn-1 exhibited a significantly longer t_(1/2)than PRD460 indicating that the target or ¹⁰Fn3 component can influencethe PK properties of Fc-¹⁰Fn3 fusion proteins. The results aresummarized in FIG. 7 and Tables 2 and 4.

TABLE 4 Pharmacokinetic properties of Adn-1 and PRD460 T-HALF V_(D) CLAUCall MRT ID (h) (mL/kg) (mL/h/kg) (h * μM) (h) Adn-1 188 81 0.35 194234 PRD460 78 104 0.92 230 74

Another experiment was performed to compare the half-life (t_(1/2)) ofFc-¹⁰Fn3 fusion proteins targeting cell-surface receptors. Thepharmacokinetics of an anti-VEGFR2 ¹⁰Fn3-Fc fusion protein (C7FLFc) andtwo other Fc-¹⁰Fn3 fusion proteins to a different cell-surface receptortarget (Adn-2 and Adn-3) were evaluated following IV administration intocynomolgus monkeys. The V_(D) and CL of Adn-2 & Adn-3 were similar toeach other but greater than observed for C7FLFc, suggesting an influenceof the target on the PK properties of Fc-¹⁰Fn3 fusion proteins. Theresults are summarized in FIG. 8 and Tables 2 and 5.

TABLE 5 Pharmacokinetic properties of C7FLFc, Adn-2 and Adn-3 DoseT-HALF V_(D) CL AUC MRT ID (mg/kg) (h) (mL/kg) (mL/h/kg) (h * μM) (h)C7FLFc 10 47 73 1 127 43 Adn-2 0.5 51 120 4.5 1.3 29 5 67 300 6.4 8.4 46Adn-3 0.5 84 150 4.2 1.4 40 5 90 210 4.3 13.9 54

Another experiment was performed to determine the bioavailability of anFc-¹⁰Fn3 fusion protein, Adn-1, in cynomolgus monkeys. Followingintravenous (IV) administration, the volume of distribution (V_(D)) ofAdn-1 was 81 mL/kg. Total body plasma clearance of Adn-1 was low (0.31mL/h/kg) and the half-life (t_(1/2)) was 188 h (FIG. 9 and Table 6).Adn-1 demonstrated subcutaneous (SC) bioavailability of 92% (FIG. 9 andTable 6).

TABLE 6 Single-dose Pharmacokinetic Parameters (mean ± SD) of Adn-1 inMonkeys. CL SC Dose T-HALF V_(D) (mL/ AUCall MRT Bioavailability Route(h) (mL/kg) h/kg) (h * μM) (h) (%) IV 188  81 0.35 194 234 n/a SC 335* —— 164 451 92 *t_(1/2) cannot accurately be determined.Pharmacokinetic Properties of Fc-¹⁰Fn3 Fusion Proteins in Mice.Materials and MethodsMouse In Vivo Study Designs

To determine the pharmacokinetic properties of various Fc-¹⁰Fn3 fusionproteins in mice, mice were dosed either IV or SC with the fusionprotein of interest and serum or plasma samples were collected atspecific time points over the course of 2-3 weeks. Samples werecollected via tail vein or retro-orbital sinus in either CPD or K₂EDTAfor plasma or in SST for serum and stored at −80° C. until analysis. Thedetails of various study designs are listed in Table 7 below.

TABLE 7 Mouse in vivo Study Designs Dose ID Mouse strain (mg/kg) Doseroute Study Duration PRD460 NCr nu 10 IV 2 weeks C57Bl/6 PRD461 NCr nu10 IV 2 weeks C57Bl/6 PRD239 NCr nu 10 IV 2 weeks PRD713 NCr nu 10 IV 2weeks Adn-1 SCID 2 IV 2 weeks SC Adn-4 SCID 0.74 IV 2 weeks SC Adn-5SCID 2 IV 2 weeks SC Adn-8 Balb/c 8 IV 2 weeks Adn-3 Balb/c 1 IV 2 weeksAdn-9 Balb/c 1 IV 2 weeks 8 IV Adn-6 C57Bl/6 2 IV 3 weeks SC Adn-7C57Bl/6 2 IV 3 weeks SC C7FLFc NCr nu 10 IV 2 weeksPharmacokinetic Properties of Fc-¹⁰Fn3 Fusion Proteins in Mice.

A series of experiments were performed in mice to evaluate the PKproperties and half-life (t_(1/2)) of various Fc-¹⁰FN3 fusion proteins.Results are summarized in FIGS. 10-14, and Tables 2, 8-10. The PKprofiles of Fc-¹⁰FN3 fusion proteins targeting soluble ligands are shownin FIG. 10 and half-lives (t_(1/2)s) are summarized in Table 2. Theresults indicate similar PK profiles for the majority of Fc-¹⁰FN3 fusionproteins examined. The half-lives ranged from 25-126 hours in mice. TwoFc-¹⁰FN3 fusion proteins exhibited a different profile from the majorityof the group and these results suggest an influence of the ¹⁰FN3component on PK.

The PK profiles of Fc-¹⁰FN3 fusion proteins targeting cell-surfacereceptors are shown in FIG. 11 and half-lives (t_(1/2)s) are summarizedin Table 2. The results indicate similar PK profiles for the majority ofFc-¹⁰FN3 fusion proteins examined. The half-lives ranged from 23-73hours in mice. Two Fc-¹⁰FN3 fusion proteins exhibited a differentprofile from the majority of the group and these results suggest aninfluence of the ¹⁰FN3 component and/or target on PK.An experiment was performed to determine whether the X-Fc or Fc-Xorientation influences Fc-¹⁰FN3 fusion protein pharmacokinetics (PK).The PK properties of PRD239 and PRD713, two Fc-¹⁰FN3 fusion proteinscreated with the same ¹⁰FN3 component were evaluated following IVadministration in nude mice. As shown in FIG. 12 and Tables 2 and 8, theorientation does not affect the PK properties in mice.

TABLE 8 Pharmacokinetic properties of two IL-23 Adnectins, PRD239 andPRD713 T-HALF V_(D) CL AUCall MRT ID Orientation (h) (mL/kg) (mL/h/kg)(h * μM) (h) PRD239 Fc-X 60.7 ± 2.9  382.5 ± 53.4 4.36 ± 0.41 29.1 ± 2.981.8 ± 3.7  PRD713 X-Fc 65.6 ± 11.8 359.1 ± 5   3.89 ± 0.8  34.2 ± 6.481.4 ± 17.1An experiment was performed to determine whether the strain of miceinfluences Fc-¹⁰FN3 fusion protein pharmacokinetics (PK). The PKproperties of PRD460 were evaluated following IV administration in nudeor C57B1/6 mice. As shown in FIG. 13 and Tables 2 and 9, the mousestrain does not affect the PK properties of Fc-¹⁰FN3 fusion proteins.

TABLE 9 Pharmacokinetic properties of PRD460 in C57Bl/6 and nude miceMouse T-HALF V_(D) CL AUCall MRT ID Strain (h) (mL/kg) (mL/h/kg) (h *μM) (h) PRD460 C57Bl/6 120.1 ± 3.5  951.3 ± 254.9 5.48 ± 1.41 23.09 ±3.48 143.1 ± 7.6  PRD460 nude 95.6 ± 12.4 941.4 ± 95.4  6.84 ± 0.2518.22 ± 0.63 121.9 ± 17.5An experiment was performed to determine whether the ¹⁰Fn3 componentaffects Fc-¹⁰FN3 fusion protein pharmacokinetics (PK). The PK propertiesof two Fc-¹⁰FN3 fusion proteins that target PCSK9, PRD460 and PRD461,were evaluated following IV administration in nude mice. As shown inFIG. 14 and Tables 2 and 10, the PCSK9 ¹⁰Fn3 component can affect the PKproperties of Fc-¹⁰FN3 fusion proteins.

TABLE 10 Pharmacokinetic properties of PRD460 and PRD461 (both PCSK9binders) T-HALF V_(D) CL AUCall MRT ID Orientation (hr) (mL/kg)(mL/hr/kg) (hr * μM) (hr) PRD460 X-Fc 95.6 ± 12.4 941.4 ± 95.4  6.84 ±0.25 18.22 ± 0.63 121.9 ± 17.5 PRD461 X-Fc 67.1 ± 11.7 3930.4 ± 1052.340.28 ± 5.1   3.33 ± 0.42 72.76 ± 8.9 

Example 10 Binding Affinity of Fc-¹⁰Fn3 Fusions Vs. Non-Fc ¹⁰Fn3Proteins

The binding properties of Fc-¹⁰Fn3 fusion proteins and non-Fc ¹⁰Fn3proteins were characterized by Surface Plasmon Resonance (SPR).Anti-human or anti-Histidine antibody was immobilized on a Biacore chip,and ¹⁰Fn3 proteins and Fc-¹⁰Fn3 fusions were captured on the chipsurface. Varying concentrations of target were placed into the flowsolution using MgCl2 (3 M) for chip regeneration between cycles. Kineticdeterminations were performed at 25° C. Evaluation of the kineticparameters was performed using the 1:1 binding algorithm on the BiacoreEvaluation software.

The results are shown in Table 11 below. In some instances, theorientation of the ¹⁰Fn3 to the Fc did not affect binding whereas inothers it did. Overall, these results show that the presence of Fc doesnot negatively affect binding affinity.

TABLE 11 Kinetic parameters for ¹⁰Fn3-Fc fusion proteins and unmodified¹⁰Fn3 proteins against captured targets. ID Target Orientation ka (1/Ms)kd (1/s) KD (nM) 1784F03 PCSK9 No Fc 1.15E+04 3.96E−04 34.46 PRD289PCSK9 X-Fc 1.20E+04 1.03E−04 8.60 PRD292 PCSK9 Fc-X 4.68E+03 1.49E−0431.82 1813E02 PCSK9 No Fc 1.75E+04 3.88E−04 22.22 PRD290 PCSK9 X-Fc1.95E+04 2.04E−04 10.47 PRD293 PCSK9 Fc-X 6.38E+03 1.72E−04 26.871922G04 PCSK9 No Fc 3.23E+04 2.10E−04 6.502 PRD 461 PCSK9 X-Fc 3.23E+041.08E−04 3.353 PRD 463 PCSK9 Fc-X 2.04E+04 8.63E−05 4.237 1459D05 PCSK9No Fc 5.56E+03 5.30E−04 95.26 PRD288 PCSK9 X-Fc 5.63E+03 3.37E−04 59.89PRD291 PCSK9 Fc-X 4.28E+03 8.23E−04 192.20 ATI-1081 PCSK9 No Fc 3.65E+042.45E−04 6.7 PRD460 PCSK9 X-Fc 3.75E+04 1.21E−04 3.29 PRD462 PCSK9 Fc-X7.33E+03 3.27E−04 44.58 C7FL VEGFR2 No Fc 2.05E+4  2.36e−4 11.5 C7FL-FcVEGFR2 X-Fc 1.07E+04 1.69E−04 15.80

Example 11 Ba/F3 Proliferation Assay

The ability of C7FL-Fc (anti-VEGFR2Fc-¹⁰Fn3) to inhibit proliferation ofBa/F3 cells was compared to inhibition by CT322 (anti-VEGFR2 ¹⁰Fn3).Ba/F3 cells stably expressing a VEGFR2 fusion protein (comprising theextracellular domain of hVEGFR2 and the intracellular domain of hEpoR)were plated in 96-well plates at 25,000 cells/well in 90 μl growth mediacontaining 15 ng/ml of VEGF-A, VEGF-C, or VEGF-D. Serial dilution ofCT322 or C7FL-Fc were prepared at 10× final concentration, and 10 μl ofCT322 or C7FL-Fc was added to each well. Plates were incubated at 37°C./5% CO2 for 48-72 hours. 20 μl of CellTiter 96® Aqueous One SolutionReagent (Promega) was added to each well, and the plates were furtherincubated for 3-4 hours at 37° C. At the end of the incubation period,absorbance was read at 490 nm using a microtiter plate reader. FIG. 15shows that C7FL-Fc can inhibit Ba/F3 proliferation equivalently toCT322. The results are summarized in Table 12.

TABLE 12 Summary of Ba/F3 proliferation assay ID IC50 (nM) RelativePotency CT-322 7.961 1 C7FL-Fc 3.374 2.36

Example 12 Evaluation of Linkers for the Generation of Fc-¹⁰Fn3 FusionProteins

Experiments were performed to evaluate the performance of 8 differentlinkers for the generation of Fc-¹⁰Fn3 fusion proteins. The fusionproteins were evaluated on four criteria: (i) protein concentration,(ii) monomer content, (iii) melting temperature, and (iv) bindingaffinity for target. Table 13 lists the different linkers chosen forthis study.

Four different ¹⁰Fn3 molecules, each specific for a different target,were fused to each linker, in the Fc-X orientation. The four different¹⁰Fn3 molecules are Adn-1, C7FL, Adn-10 and 2013. In total, 32 differentFc fusion molecules were generated and analyzed.

TABLE 13 Linkers SEQ Num- ID ber Linker Length Description NO. 1 QPDEP 5 Derived from 81 human CH2—CH3 link; R→D 2 AGGGGSG  7 Standard 37linker in Fc-X Adnectin fusions. 3 PVPPPPP  7 IgA2 hinge, 82 rigid 4(ED)₅E 11 Synthetic, 83 solubilizing, flexible 5 DLPQETLEEETPGA 14Derived from 84 membrane IgA tail sequence 6 VPSTPPTPSPST 12 IgA1 hinge85 short 7 ELQLEESAAEAQEGELE 17 Derived from 86 membrane IgG1tail sequence (D→E) 8 ESPKAQASSVPTAQPQAE 18 IgD hinge 1st 87 exon longHigh-Throughput Mammalian Expressed Protein (HMEP) Analysis

Expression constructs encoding the 32 Fc-¹⁰Fn3 fusion proteins weretransfected into 4 ml of HEK-293-6E culture using 24 deep-well platesand incubated and incubated at 37° C. Five days post-transfection, thecells were lysed and protein was purified using Protein A HP Multitrap.The resulting protein preparation was evaluated for protein yield usinga BCA Protein assay with SGE (control Adnectin™) as the proteinstandard.

FIG. 18 is a graph summarizing the average yield per transfection volumeof each Fc-¹⁰Fn3 fusion series. Diamonds represent the And-1 series,squares represent the Fc-C7FL series, triangles represent the Adn-10series, and crosses represent the Fc-2013 series. Overall, the Adn-1series had the highest average yield per transfection volume.

Size exclusion chromatography (SEC) was performed on the Fc-¹⁰Fn3 fusionproteins resulting from the HMEP. SEC was performed using a Superdex 2005/150 or Superdex 75 5/150 column (GE Healthcare) on an Agilent 1100 or1200 HPLC system with UV detection at A₂₁₄ nm and A₂₈₀ nm and withfluorescence detection (excitation=280 nm, emission=350 nm). A buffer of100 mM sodium sulfate, 100 mM sodium phosphate, 150 mM sodium chloride,pH 6.8 at appropriate flow rate of the SEC column employed. Gelfiltration standards (Bio-Rad Laboratories, Hercules, Calif.) were usedfor molecular weight calibration.

FIG. 19 is a graph summarizing the monomer score of each Fc-¹⁰Fn3 fusionseries. Labels are the same as in FIG. 18. Results show that Fc-¹⁰Fn3fusions with linker 7 have high percent monomer score.

Midscale Expressed Protein Analysis

The Adn-1 linker series was chosen for midscale analysis. Expressionconstructs encoding the Adn-1 linker series were transfected into 175 mlof HEK-293-6E. Five days post-transfection, the cells were lysed andprotein was purified using Protein A purification on an AKTA 100. Theresulting protein preparation was evaluated for protein yield using aBCA Protein assay with SGE (control Adnectin™) as the protein standard.

FIG. 20 is a graph summarizing the average yield for the Adn-1 linkerseries. Results show that yield is high for most Adn-1 fusions.

SEC analysis of the midscale purified Adn-1 fusions demonstrated thatmost Adn-1 fusions have high monomer content. FIG. 21 is a graphsummarizing the monomer score for each of the Adn-1 fusions.

Liquid chromatography-mass spectrometry (LC-MS) was performed on themidscale purified Fc-¹⁰Fn3 fusion proteins. FIG. 22 summarizes the LC-MSresults, which confirms the identities of seven of the tested Adn-1fusions. Representative LC-MS plots for fusions with linkers 5 and 7 areshown.

The melting temperatures of the midscale purified Fc-¹⁰Fn3 fusionproteins were measured by differential scanning calorimetry (DSC). A 1mg/ml solution of each of the Fc-¹⁰Fn3 fusion protein preparation wasscanned in a N-DSC II calorimeter (calorimetry Sciences Corp) by rampingthe temperature from 5° C. to 95° C. at a rate of 1 degree per minuteunder 3 atm pressure. The data was analyzed vs. a control run of theappropriate buffer using a best fit using Orgin Software (OrginLabCorp). FIG. 23 shows the melting temperatures for each of the Adn-1fusions compared to control, which in this experiment are the CH2 andCH3 domains of Fc. Overall, the Adn-1 fusions have melting temperaturescomparable to that of unmodified Adn-1 (no-Fc), which was previouslydetermined to be 57° C.

The binding characteristics of each of the midscale purified Fc-¹⁰Fn3fusion proteins to target were characterized by Surface PlasmonResonance (SPR). FIG. 24 summarizes the binding properties of the Adn-1series to immobilized target. Results show that all Adn-1 fusions retainbinding affinity to target.

Example 13 Immunogenicity Characterization of Linkers Used for theGeneration of Fc-¹⁰Fn3 Fusion Proteins

The adaptive immune response is initiated by the processing anddigestion of an internalized protein by an antigen-presenting cell(APC), such as a dendritic cell. The APC clips the internalized proteininto short peptides and then displays the peptides on its surface MHCClass II molecules. The peptide binding site of the MHC Class IImolecule is long and narrow, like a hot-dog bun, and holds its peptidein an extended format, with room for nine amino acids in the primarybinding site (and generally allows for short tails on either side of thepeptide). Certain pockets in the MHC binding site are dominant indetermining peptide binding. These pockets correspond to amino acidpositions 1, 4, 6, and 9 in the anchored portion of the 9-mer peptide. Apeptide that has favorable side chains at each of these four positionswill in general bind to HLA (an MHC Class II molecule) well.

Position 1 is thought to be the most important ‘anchor residue’ involvedin binding between the peptide and the HLA molecule. Position 1generally favors a hydrophobic side chain—thus, 9-mers that often bindHLA are initiated with V, I, L, M, F, Y, or W. The other positions aremuch more variable, with different HLA alleles favoring different setsof amino acids at each site.

HLA binding may be predicted in silico, for example, using EpiMatrix.EpiMatrix is a proprietary computer algorithm developed by EpiVax, whichis used to screen protein sequences for the presence of putative HLAbinding motifs. Input sequences are parsed into overlapping 9-mer frameswhere each frame overlaps the last by 8 amino acids. Each of theresulting frames is then scored for predicted binding affinity withrespect to a panel of eight common Class II HLA alleles (DRB1*0101,DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, andDRB1*1501). Raw scores are normalized against the scores of a largesample of randomly generated peptides. The resulting “Z” score isreported. Any 9-mer peptide with an EpiMatrix Z-score in excess of 1.64is considered a putative HLA binding motif.

The immunogenicity of linkers used to generate Fc-¹⁰Fn3 fusion proteinswas predicted using the above described in silico method. Table 14 liststhe amino acid sequences of the linkers analyzed (highlighted in gray)plus flanking regions, in this case the C-terminus of IgG1 Fc and theN-terminus of the ¹⁰Fn3 domain.

Table 15 shows the EpiMatrix score for each of the linkers analyzed. Allscores are very low (negative numbers in the “EpiMatrix CLUSTER SCORE”column”), indicating that the linkers are predicted to have very lowimmunogenicity.

TABLE 14 Linker sequences analyzed for immunogenicity SEQ SEQ ID NO.Linker ID NO  Sequence (highlighted portion) Fc_linker_1 133

81 Fc_linker_2 134

37 Fc_linker_3 135

82 Fc_linker_4 136

83 Fc_linker_5 137

84 Fc_linker_6 138

85 Fc_linker_7 139

86 Fc_linker_8 140

87 Fc_linker_9 141

88 Fc_linker_10 142

132  Fc_linker_11 143

86 Fc_linker_12 144

90 Fc_linker_13 145

91 Fc_linker_14 146

92 Fc_linker_15 147

93 Fc_linker_16 148

94 Fc_linker_17 149

95 Fc_linker_18 150

96 Fc_linker_19 151

97 Fc_linker_20 152

98

TABLE 15 Linker EpiMatrix results Cluster Address Input (w/ SequenceFLANKS) Cluster Sequence (SEQ ID NO) FC_LINKER_1 1-21QKSLSLSPQPDEPGVSGVPRD (133) FC_LINKER_10 1-23QKSLSLSPELQLEESGVSDVPRD (142) FC_LINKER_11 1-33QKSLSLSPELQLEESAAEAQEGELEGVSDVPRD (143) FC_LINKER_12 1-29QKSLSLSPVPSTPPTPSPSTGGVSDVPRD (144) FC_LINKER_13 1-36QKSLSLSPVPSTPPTPSPSTPPTPSPSGGVSDVPRD (145) FC_LINKER_14 1-32QKSLSLSPGRGGEEKKKEKEKEEGGVSDVPRD (146) FC_LINKER_15 1-41QKSLSLSPGRGGEEKKKEKEKEEQEERETKTPGGVSDVPRD (147) FC_LINKER_16 1-26QKSLSLSPESPKAQASSGGVSDVPRD (148) FC_LINKER_17 1-35QKSLSLSPESPKAQASSVPTAQPQAEGGVSDVPRD (149) FC_LINKER_18 1-39QKSLSLSPSVEEKKKEKEKEEQEERETKTPGGVSDVPRD (150) FC_LINKER_19 1-40QKSLSLSPPSVEEKKKEKEKEEQEERETKTPGGVSDVPRD (151) FC_LINKER_2 1-23QKSLSLSPAGGGGSGGVSDVPRD (134) FC_LINKER_20 1-40QKSLSLSPGSVEEKKKEKEKEEQEERETKTPGGVSDVPRD (152) FC_LINKER_3 1-23QKSLSLSPPVPPPPPGVSDVPRD (135) FC_LINKER_4 1-27QKSLSLSPEDEDEDEDEDEGVSDVPRD (136) FC_LINKER_5 1-30QKSLSLSPDLPQETLEEETPGAGVSDVPRD (137) FC_LINKER_6 1-28QKSLSLSPVPSTPPTPSPSTGVSDVPRD (138) FC_LINKER_7 1-33QKSLSLSPEPQLEESAAEAQEGELEGVSDVPRD (139) FC_LINKER_8 1-34QKSLSLSPESPKAQASSVPTAQPQAEGVSDVPRD (140) FC_LINKER_9 1-22QKSLSLSPPAVPPPGVSDVPRD (141) tReg EpiMatrix Adjusted EpiMatrix CLUSTERCLUSTER HITS SCORE Score Input Hydro- (w/o (w/o (w/o Sequence phobicityFLANKS) FLANKS) FLANKS) FC_LINKER_1 −1.11 1  −9.02  −9.02 FC_LINKER_10−0.73 4  −4.53  −4.53 FC_LINKER_11 −0.78 4 −13.79 −13.79 FC_LINKER_12−0.63 1 −15.57 −15.57 FC_LINKER_13 −0.75 1 −21.33 −21.33 FC_LINKER_14−1.76 1 −17.88 −17.88 FC_LINKER_15 −1.99 1 −25.30 −25.30 FC_LINKER_16−0.82 0 −14.83 −14.83 FC_LINKER_17 −0.80 0 −22.25 −22.25 FC_LINKER_18−1.86 4 −18.19 −18.19 FC_LINKER_19 −1.86 2 −22.40 −22.40 FC_LINKER_2−0.47 1 −10.68 −10.68 FC_LINKER_20 −1.83 3 −20.68 −20.68 FC_LINKER_3−0.66 0 −12.36 −12.36 FC_LINKER_4 −1.79 0 −15.66 −15.66 FC_LINKER_5−0.88 1 −14.60 −14.60 FC_LINKER_6 −0.64 1 −14.74 −14.74 FC_LINKER_7−0.78 4 −13.79 −13.79 FC_LINKER_8 −0.81 0 −21.42 −21.42 FC_LINKER_9−0.46 0 −11.54 −11.54

Example 14 Immunogenicity of Fc-¹⁰Fn3 Fusion Protein in CynomolgusMonkeys

Experiments were performed to examine whether fusion to a cynomolgus Fccould decrease the immunogenicity of ¹⁰Fn3 proteins. In theseexperiments, the immunogenicity response in cynomolgus monkeys inducedby anti-IL23 ¹⁰Fn3-Fc (1571G04-Fc) was compared to the immunogenicityresponse induced by anti-IL23 ¹⁰Fn3-PEG (1571G04-PEG). These twomolecules share the same ¹⁰Fn3 portion.

Three cynomolgus monkeys were injected i.v. with 3 mg/kg of 1571G04-PEGor 1571G04-Fc on Days 1, 8 and 15. Plasma samples were collected on Days1, 8, 15 prior to each injection as well as at 168, 240, 336, 408 and504 hours after the 3^(rd) dose. Plasma was analyzed for anti-adnectinantibodies in a typical ELISA assay. In short, 1571G04-PEG or 1571G04-Fcwas adsorbed to microtiter plates and anti-drug antibodies in plasmasamples are captured and detected with rabbit anti-human IgG-HRPconjugated antibodies. A positive response is defined as greater thantwice the background level observed at the predose 1 time point for eachanimal.

As shown in FIG. 27, 1571G04-PEG induced a significant anti-¹⁰Fn3 IgGresponse after three weekly i.v. injections of 3 mg/kg. In contrast andshown in FIG. 28, the 1571G04-Fc molecule induced very little anti-¹⁰Fn3IgG response, such that we did not see an increase in antibodies at anytime-point analyzed.

These results suggest that fusion of ¹⁰Fn3 proteins to a cynomolgus Fccan decrease the inherent immunogenicity of ¹⁰Fn3 proteins in cynomolgusmonkeys, suggesting that a human Fc fused to ¹⁰Fn3 proteins may decreasethe immunogenicity of ¹⁰Fn3 proteins in humans.

Example 15 STAT3 Phosphorylation on Kit225 Cells Method

Parham et al. (A receptor for the heterodimeric cytokine IL-23 iscomposed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R.J. Immunol. 2002 Jun. 1; 168(11):5699-708) cloned the IL-23R from thehuman IL-2 dependent T-cell line, Kit225. These cells have beencharacterized for expression of both IL-12RB1 and 1L-23R by FACSanalysis and responded to IL-23 by stimulation of pSTAT3 and to IL-12 bystimulation of pSTAT4. Kit225 cells were seeded into 96 well plates andquiesced in the absence of FBS and IL-2 for 3 hrs at 37° C. Followingthis incubation, 10 pM human recombinant IL-23 (or IL-23 preincubatedwith antagonist for 1 hr) was applied and the cells returned to theincubator for 15 minutes at 37° C. to stimulate the phosphorylation ofSTAT3 (abbreviated as p-STAT3). Each condition was assayed in duplicatein 96-well plates. Stimulation was stopped by placing the cells on iceand addition of ice-cold PBS. Finally, the cells were pelleted and lysedfollowing standard protocols and pSTAT3 production detected by ELISA.

Results

Stimulation of IL23R by IL23 in Kit225 cells was assessed by measuringpSTAT3. This stimulation was effectively inhibited by the base anti-IL23Adnectin clone 1571 G04 resulting in an IC₅₀ of 86.1±8.1 pM. IL23inhibition by the 1571G04-Fc fusion protein was comparable to theunformatted Adnectin, yielding an IC₅₀ of 153±19 μM. The alternativeorientation of Fc-1571G04 resulted in a significant loss of activity inthis assay (IC₅₀=692±159 pM). These results are summarized in Table 16.

TABLE 16 Stat3 phosphorylation in Kit225 cells. Clone pSTAT3 IC50 (pM)1571G04 86.1 ± 8.1 (n = 2) PRD239 (Fc-1571G04)  692 ± 159 (n = 2) PRD713(1571G04-Fc)  153 ± 19 (n = 2)

Example 16 Amino Acid Sequences of Fusion Proteins Used in the Examples

PRD289: (SEQ ID NO: 122)GVSDVPRDLEVVAATPTSLLISWRPPIHAYGYYRITYGETGGNSPVQEFTVPIVEGTATISGLKPGVDYTITVYAVEYTFKHSGYYHRPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.PRD289 has the following hinge: EPKSSGSTHTCPPCPAPELLGGSS(SEQ ID NO: 26) and a human IgG1 Fc. PRD292: (SEQ ID NO: 123)EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGVSDVPRDLEVVAATPTSLLISWRPPIHAYGYYRITYGETGGNSPVQEFTVPIVEGTATISGLKPGVDYTITVYAVEYTFKHSGYYHRPISINYRTEIPRD292 has the following hinge: EPKSSGSTHTCPPCPAPELLGGSS andthe following linker: AGGGGSG, and a human IgG1 Fc. PRD290:(SEQ ID NO: 124)GVSDVPRDLEVVAATPTSLLISWSPPANGYGYYRITYGETGGNSPVQEFTVPVGRGTATISGLKPGVDYTITVYAVEYTYKGSGYYHRPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPRD290 has the following hinge: EPKSSGSTHTCPPCPAPELLGGSSand a human IgG1 Fc. PRD293: (SEQ ID NO: 125)EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGVSDVPRDLEVVAATPTSLLISWSPPANGYGYYRITYGETGGNSPVQEFTVPVGRGTATISGLKPGVDYTITVYAVEYTYKGSGYYHRPISINYRTEIPRD293 has the following hinge: EPKSSGSTHTCPPCPAPELLGGSSand the following linker: AGGGGSG and a human IgG1 Fc. PRD713:(SEQ ID NO: 126)GVSDVPRDLEVVAATPTSLLISWGHYPLHVRYYRITYGETGGNSPVQEFTVPPRSHTATISGLKPGVDYTITVYAVTYYAQENYKEIPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPRD713 has the following hinge: EPKSSGSTHTCPPCPAPELLGGSSand a human IgG1 Fc. PRD239: (SEQ ID NO: 127)EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGVSDVPRDLEVVAATPTSLLISWGHYPLHVRYYRITYGETGGNSPVQEFTVPPRSHTATISGLKPGVDYTITVYAVTYYAQENYKEIPISINYRTEASPRD239 has the following hinge: EPKSSGSTHTCPPCPAPELLGGSSand the following linker AGGGGSG and a human IgG1 Fc. C7FL-Fc (PRD1309):(SEQ ID NO: 128)GSVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK C7FL-Fc (PRD1309) has the following hinge:EPKSSDKTHTCPPCPAPELLGGSS and a human IgG1 Fc. C7FL-Fc (PRD1308):(SEQ ID NO: 129)GSVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC7FL-Fc (PRD1308) has the following hinge:EPKSSDKTHTCPPCPAPELLGGPS and a human IgG1 Fc.PRD461 is a fusion protein comprising an Fc linked to the anti-PCSK9Adnectin 2013E01, whose sequence is provided in WO2011/130354. The aminoacid sequence for the anti anti-PCSK9 adnectins 1784F03 and 1813E02 areprovided in WO2011/130354. The amino acid sequence of the anti-IL-23adnectin 1571G04 is provided in WO2011/103105.

INCORPORATION BY REFERENCE

All documents and references described herein are individuallyincorporated by reference to into this document to the same extent as ifthere were written in this document in full or in part.

The invention claimed is:
 1. A polypeptide comprising an immunoglobulin IgG Fc domain and a heterologous polypeptide, wherein the heterologous polypeptide is fused to the N-terminus or the C-terminus of the Fc domain by a polypeptide linker, wherein the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-54, 63-65 and
 84. 2. The polypeptide of claim 1, wherein the polypeptide linker comprises SEQ ID NO:
 84. 3. The polypeptide of claim 1, wherein the heterologous polypeptide is fused to the C-terminus of the Fc domain.
 4. The polypeptide of claim 1, wherein the heterologous polypeptide is fused to the N-terminus of the Fc domain.
 5. The polypeptide of claim 1, wherein the heterologous polypeptide comprises a ¹⁰Fn3 domain.
 6. The polypeptide of claim 1, wherein the immunoglobulin Fc domain comprises a hinge or a portion thereof.
 7. The polypeptide of claim 1, wherein the polypeptide linker consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-54, 63-65 and
 84. 